Bidirectional effect of serum amyloid A on plaque stability Bo Li a,b, Bei-bei Luo a,b, Wei-dong Qin a,b, Hui Liu a,b, Yan-fei Xia a,b, Tong-xiang Liu c, Jing-tian Li d, Ming-xiang Zhang a,b, Yun Zhang a,b, Cheng Zhang a,b, Feng-shuang An a,b,⁎ a b c d
Key Laboratory of Cardiovascular Remodeling and Function Research Chinese Ministry of Education and Chinese Ministry of Public Health, Ji'nan 250012, PR China Department of Cardiology, Qilu Hospital of Shandong University, Ji'nan 250012, PR China People's Hospital of Weifang, Weifang, Shandong, PR China Afﬁliated Hospital of Weifang Medical University, Weifang, Shandong, PR China
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Article history: Received 15 February 2014 Accepted 29 March 2014 Available online 6 April 2014 Keywords: Atherosclerosis Plaque stability Serum amyloid A Collagen
The rupture of atherosclerotic plaques can lead to atherothrombotic events. The imbalance between collagen degradation and collagen synthesis is one of the most important determinants of plaque vulnerability [1,2]. Serum amyloid A protein (SAA) plays an important role in the host defense system, through restricting tissue damage and promoting tissue healing [3,4]. SAA has been found in many cell types in atherosclerotic lesions, including endothelial cells, smooth muscle cells, monocytes and macrophages [5,6]. It has been demonstrated that the plasma concentration of SAA may be a marker of acute coronary syndromes  and stable coronary artery disease . SAA could affect the clearance of cholesterol in the atherosclerotic lesion . SAA could also promote the chemotaxis, adhesion and migration of monocytes/macrophages . SAA could induced the expression of various cytokines, such as TNF-α, IL-1β, IL-6, IL-8, MCP-1 and Lp-PLA2 [11,12]. The effects of SAA above have been considered as the main cause of plaque vulnerability [13,14]. However, SAA could also stimulate proteoglycan synthesis and increase plaque stability by increasing TGF-β expression . Moreover, SAA might inhibit platelet aggregation  and modulate platelet adhesion at vascular injury sites [17,18]. Thus, it appeared that SAA could increase plaque stability on the other side. In the present study, a series of experiments in vivo and in vitro were designed and performed to investigate the exact role of SAA on plaque disruption. Male ApoE−/− mice (n = 80, 6 weeks of age) were purchased from Peking University (Beijing, China). Just as previously described , after a chow diet (5% fat and no added cholesterol) for 2 weeks, mice were received a constrictive silastic tube (0.30 mm inner diameter, 0.50 mm outer diameter, and 2 mm long), which was placed around the left common carotid artery, followed with high-fat diet for 8 weeks. After that, the mice were randomly divided into 4 groups: control group (n = 20, locally injection of 0.2 ml physiological saline in left carotid artery region), lenti-null group (n = 20, locally injection of 1 × 109 TU null lenti-virus in left carotid artery region), low-lenti-SAA1 group (n = 20, locally injection with a mixture of 1 × 107 TU lenti-SAA1 and 1 × 109 TU null lenti-virus in left carotid artery region) and high-lenti-SAA1 group (n = 20,
locally injection of 1 × 109 TU lenti-virus containing SAA1 in left carotid artery region). At last another 4 weeks high-fat diet was feeding. As shown in Fig. 1, all of the immunohistochemistry, Western blot and real-time PCR indicated that the expression of SAA1 was signiﬁcantly up-regulated after transfection, and directly proportional to the dosage of the lentivirus, while transfection with lenti-null had no effect on the expression of SAA1 compared with control group. In order to calculate the vulnerable index precisely, the component areas of lipids, macrophages, SMCs and collagen were quantitated. The vulnerable index was calculated by the formula as follow: the relative positive staining areas of (macrophages % + lipid %)/the relative positive staining areas of (α-SMCs% + collagen%) . As shown in Fig. 1, the relative contents of lipids of low-lentiSAA1 transfection group was slightly elevated compared with control group (P N 0.05), but it was signiﬁcantly elevated in highlenti-SAA1 transfection group (P b 0.05). The relative contents of macrophages of both low-lenti-SAA1 group and high-lenti-SAA1 group were signiﬁcantly elevated compared with control group (P b 0.05). However, the relative contents of collagen in the lowlenti-SAA1 group was greatly elevated compared with control group (P b 0.05), inconceivablely, it was decreased in the high-lenti-SAA1 group compared with low-dose SAA1 transfection group (P b 0.05), and there was no difference between the high-lenti-SAA1 group and control groups (P N 0.05); while there was no difference of the relative contents of SMCs among the four groups (P N 0.05). The vulnerability index of the low-dose SAA1 group was lower than control group (P b 0.05). However, the vulnerability index of highlenti-SAA1 group was evidently higher than the low-lenti-SAA1 group (P b 0.05) and control groups (P b 0.05). These results demonstrated that lenti-virus transfection of SAA1 changed the composition and vulnerability of the carotid plaque in a bidirectional manner. In order to disclose the potential mechanisms, we further investigated the detailed effects of SAA1 on Collagen I and Collagen III expressions in vivo and in vitro. We ﬁrstly detected the expression of Collagen I and immunohistochemistry showed that low-lentiSAA1 could up-regulate both Collagen I and Collagen III compared with control group (P b 0.05). But high-lenti-SAA1 reduced the expression of collagen compared with low-dose lenti-SAA1 group (P b 0.05) and there was no difference between high-lenti-SAA1 and control group (P N 0.05). Western blot exhibited a similar result (Fig. 2C and D). Following, the effects of SAA1 on collagen expression were evaluated in mice and human SMCs. Various concentrations of recombinant human apo-SAA1 (0, 0.01, 0.1, 1 and 10 μg/ml) were used to stimulate cells for 24 h. As shown in Fig. 2E and F, SAA1 increased collagen I and collagen III in human and mice SMCs in a dose-dependent manner. The results demonstrated that SAA1 affected at the concentration of 1 μg/ml (mice collagen I: P b 0.05; human collagen I: P b 0.05; mice collagen III: P b 0.05; human collagen III: P b 0.05) and reached the maximal effect at 10 μg/ml (mice collagen I: P b 0.01; human collagen I: P b 0.05; mice collagen III: P b 0.01; human collagen III: P b 0.01). Then, 1 μg/ml SAA1 was chosen to stimulate mice and human SMCs for different time (1 h, 3 h, 6 h, 12 h and 24 h). As shown in Fig. 2E and F, SAA1 increased
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collagen I and collagen III at 6 h (mice collagen I: P b 0.05; human collagen I: P b 0.05; mice collagen III: P b 0.05; human collagen III: P b 0.05) and peaked at 24 h (mice collagen I: P b 0.01; human collagen I: P b 0.05; mice collagen III: P b 0.01; human collagen III: P b 0.01). At last, human SMCs were stimulated with 1 μg/ml of SAA1 for 24 h, and collagen I and collagen III expressions were detected by green ﬂuorescent dye. The immunoﬂuorescent showed that SAA1 increased collagen I and collagen III accumulation in the plasma (Fig. 2G). These results demonstrated that SAA1 had the ability to induce collagen I and collagen III expressions in vitro, but had a bidirectional effect in vivo. MMP-1 and MMP-8 are the major collagenases which can efﬁciently degrade collagen I and collagen III. In order to investigate the mechanism in which low-lenti-SAA1 increased plaque stability while high-lenti-SAA1 lost the ability, MMP-1 and MMP-8 were investigated both in vivo and in vitro, respectively. In ApoE−/− mice, the expression of MMP-1 in the carotid plaques as assessed by immunohistochemistry was unchanged both in the low-lenti-SAA1 group and high-lenti-SAA1 group compared with the control group (P N 0.05, Fig. 3A and B). Whereas, although the expression of MMP-8 was still unchanged in the low-lenti-SAA1 (P N 0.05, Fig. 3A and B), it was dramatically enhanced in the high-lenti-SAA1 group (P b 0.01, Fig. 3A and B) compared with the control group. For further investigation, RAW264.7 macrophages were cultured and treated with gradient concentrations of SAA1 (0, 0.01, 0.1,1, 5 and 10 μg/ml). After SAA1 treatment for 12 h, MMP-1 and MMP-8 were detected, and the results indicated that there was no signiﬁcant change of MMP-1 (P N 0.05, Fig. 3D and E), while MMP-8 was up-regulated when the SAA1 reached 5 μg/ml (P b 0.01, Fig. 3D and E) and 10 μg/ml (P b 0.01, Fig. 3D and E). The Western blot also validated the results, as shown in Fig. 3C. Subsequently, the expression of MMP-2 and MMP-9 were also detected, which also contributed to promote plaque destabilization and disruption. As shown in Fig. 3D and E, SAA1 displayed its strong ability to increase MMP-2 and MMP-9 from the concentration of 1 μg/ml (P b 0.05, Fig. 3D and E), and reached the peak at 5 and 10 μg/ml (P b 0.01, Fig. 3D and E). All the data above suggested that SAA1 could dramatically improve the expression of MMP-8, MMP-2 and MMP-9 in a dose-dependent manner, but had no effect on MMP-1. In conclusion, SAA1 dose- and time-dependently induced collagen I and collagen III expressions, which was beneﬁcial for the plaque stability. However, high-dose SAA1 could enhance MMP-8, MMP-2 and MMP-9, which was mediated the plaque degradation. Thus, the effect of SAA1 on plaque stability was bi-directional. It might be a new approach to protect atherosclerotic plaque through regulating the locally expression level of SAA1. And further studies should be performed to validate the conclusion and clarify the underlying molecular signaling mechanisms. In the present study, we had several novel ﬁndings: (1) SAA1 played bidirectional roles in plaque stability. Low-dose lenti-SAA1 infusion (1 × 107 TU) signiﬁcantly enhanced plaque stability, while high-dose lenti-SAA1 infusion (1 × 109 TU) lost its protective effect; (2) SAA1 exerted its plaque protective effect by improving collagen synthesis; (3) SSA1 increased collagen synthesis via Smad2/3 pathway, but independent
of TGF-β1 and MAPKs; (4) SAA1 increased plaque vulnerability by increasing MMPs expression and collagen degradation. To our knowledge, the present study provided the bidirectional effects of SAA1 on plaque stability and the positive effects of SAA1 on collagen expression for the ﬁrst time. This study was supported by the National 973 Basic Research program (2009CB521904) and Natural Science Foundation of China (no. 81370325). References  Shah PK, Falk E, Badimon JJ, et al. Human monocyte-derived macrophages induce collagen breakdown in ﬁbrous caps of atherosclerotic plaques. Potential role of matrix-degrading metalloproteinases and implications for plaque rupture. Circulation 1995;92:1565–9.  Rekhter MD. Collagen synthesis in atherosclerosis: too much and not enough. Cardiovasc Res 1999;41:376–84.  Steel DM, Whitehead AS. The major acute phase reactants: C-reactive protein, serum amyloid P component and serum amyloid A protein. Immunol Today 1994;15:81–8.  Sammalkorpi KT, Valtonen VV, Maury CP. Lipoproteins and acute phase response during acute infection. Interrelationships between C-reactive protein and serum amyloid-A protein and lipoproteins. Ann Med 1990;22:397–401.  Meek RL, Urieli-Shoval S, Benditt EP. Expression of apolipoprotein serum amyloid A mRNA in human atherosclerotic lesions and cultured vascular cells: implications for serum amyloid A function. Proc Natl Acad Sci U S A 1994;91:3186–90.  Urieli-Shoval S, Meek RL, Hanson RH, Eriksen N, Benditt EP. Human serum amyloid A genes are expressed in monocyte/macrophage cell lines. Am J Pathol 1994;145:650–60.  Morrow DA, Rifai N, Antman EM, et al. Serum amyloid A predicts early mortality in acute coronary syndromes: a TIMI 11A substudy. J Am Coll Cardiol 2000;35:358–62.  Ogasawara K, Mashiba S, Wada Y, et al. A serum amyloid A and LDL complex as a new prognostic marker in stable coronary artery disease. Atherosclerosis 2004;174:349–56.  Urieli-Shoval S, Linke RP, Matzner Y. Expression and function of serum amyloid A, a major acute-phase protein, in normal and disease states. Curr Opin Hematol 2000;7:64–9.  Badolato R, Wang JM, Murphy WJ, et al. Serum amyloid A is a chemoattractant: induction of migration, adhesion, and tissue inﬁltration of monocytes and polymorphonuclear leukocytes. J Exp Med 1994;180:203–9.  Song C, Hsu K, Yamen E, et al. Serum amyloid A induction of cytokines in monocytes/macrophages and lymphocytes. Atherosclerosis 2009;207:374–83.  Li B, Dong Z, Liu H, et al. Serum amyloid A stimulates lipoprotein-associated phospholipase A2 expression in vitro and in vivo. Atherosclerosis 2013;228:370–9.  Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest 1994;94:2493–503.  Sukhova GK, Schonbeck U, Rabkin E, et al. Evidence for increased collagenolysis by interstitial collagenases-1 and -3 in vulnerable human atheromatous plaques. Circulation 1999;99:2503–9.  Wilson PG, Thompson JC, Webb NR, de Beer FC, King VL, Tannock LR. Serum amyloid A, but not C-reactive protein, stimulates vascular proteoglycan synthesis in a pro-atherogenic manner. Am J Pathol 2008;173:1902–10.  Zimlichman S, Danon A, Nathan I, Mozes G, Shainkin-Kestenbaum R. Serum amyloid A, an acute phase protein, inhibits platelet activation. J Lab Clin Med 1990;116:180–6.  Sayinalp N, Haznedaroglu IC, Buyukasik Y, et al. Protein C inhibitor and serum amyloid A in immune thrombocytopaenic purpura. J Int Med Res 2004;32:62–5.  Urieli-Shoval S, Shubinsky G, Linke RP, Fridkin M, Tabi I, Matzner Y. Adhesion of human platelets to serum amyloid A. Blood 2002;99:1224–9.  Li JJ, Meng X, Si HP, et al. Hepcidin destabilizes atherosclerotic plaque via overactivating macrophages after erythrophagocytosis. Arterioscler Thromb Vasc Biol 2012;32:1158–66.
Fig. 1. Expression of SAA1 in 3 groups of mice and effects of different doses of lenti-SAA1 transfection played on the plaque composition.A, Staining for the carotid plaques SAA1, lipids (red), macrophages (brown), SMCs (brown) and collagens (green) in 4 treatment groups were shown. B, Quantitative analysis of the results in A (n = 20 in each group). C, Western blot was used to detect SAA1 expression in the carotid plaques of the 3 treatment groups. D, Real-time PCR was used to detect SAA1 mRNA expression in the carotid plaques of the 4 treatment groups. E, Quantitative analysis of the results in A (n = 20 in each group). F, Vulnerability index were analyzed in 4 treatment groups. *P b 0.05 versus control group, **P b 0.01 versus control group; #P b 0.05 versus low-lenti-SAA1 group, ##P b 0.01 versus low-lenti-SAA1 group.
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Fig. 2. Effects of SAA1 played on collagen I and collagen III expressions in vivo and in vitro.A, Immunochemical staining of collagen I and III in the plaques in 4 treatment groups were shown. The positive staining areas were in brown. B, Quantitative analysis of the results in A (n = 20 in each group). C and D, Western blot was used to detect collagen I and III expressions in the carotid plaques of the 4 treatment groups. E and F, after SMCs were stimulated for 24 h with SAA1 of different concentration gradients (0, 0.01, 0.1, 1, 10 μg/ml) or stimulated with 1 μg/ml SAA1 for different times (0, 1, 3, 6, 12, 24 h), Western blot was used to detect the expression of collagen I and III at protein level. G, SMCs were incubated with 1 μg/ml SAA1 for 24 h. After that, the cells were used for immune- ﬂuorescence staining to observe the role of SAA1 on collagen I and III (green) expressions (magniﬁcation 400×). *P b 0.05 versus control group; **P b 0.01 versus control group; #P b 0.05 versus low-lenti-SAA1 group, ##P b 0.01 versus low-lenti-SAA1 group.
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Fig. 3. Effects of SAA1 played on the expression of MMPs.A, Immunochemical staining of MMP-1 and MMP-8 in the plaques in 4 treatment groups were shown. The positive staining areas were in brown. B, Quantitative analysis of the results in A (n = 20 in each group). C, RAW264.7 cells were incubated with 1 and 10 μg/ml SAA1 for 24 h. After that, the cells were used for immune-ﬂuorescence staining to observe the role of SAA1 on MMP-1 and MMP-8 (green) expression (magniﬁcation 400 ×). D and E, After RAW264.7 cells were stimulated for 24 h with SAA1 of different concentration gradients (0, 0.01, 0.1, 1, 5, 10 μg/ml), Western blot was used to detect the expression of MMP-1, MMP-8, MMP-2 and MMP-9 at protein level. *P b 0.05 versus control group; **P b 0.01 versus control group.