A brief elevation of serum amyloid A is sufficient to increase atherosclerosis

Abstract

Serum amyloid A (SAA) has a number of proatherogenic effects including induction of vascular proteoglycans. Chronically elevated SAA was recently shown to increase atherosclerosis in mice. The purpose of this study was to determine whether a brief increase in SAA similarly increased atherosclerosis in a murine model. The recombination activating gene 1-deficient (rag1(-/-)) × apolipoprotein E-deficient (apoe(-/-)) and apoe(-/-) male mice were injected, multiple times or just once respectively, with an adenoviral vector encoding human SAA1 (ad-SAA); the injected mice and controls were maintained on chow for 12-16 weeks. Mice receiving multiple injections of ad-SAA, in which SAA elevation was sustained, had increased atherosclerosis compared with controls. Strikingly, mice receiving only a single injection of ad-SAA, in which SAA was only briefly elevated, also had increased atherosclerosis compared with controls. Using in vitro studies, we demonstrate that SAA treatment leads to increased LDL retention, and that prevention of transforming growth factor beta (TGF-β) signaling prevents SAA-induced increases in LDL retention and SAA-induced increases in vascular biglycan content. We propose that SAA increases atherosclerosis development via induction of TGF-β, increased vascular biglycan content, and increased LDL retention. These data suggest that even short-term inflammation with concomitant increase in SAA may increase the risk of developing CVD.

Keywords: apolipoproteins; cardiovascular disease; extracellular matrix; free-form: biglycan; lipoproteins; proteoglycans; transforming growth factor beta; vascular biology.

Fig. 1.

SAA-stimulated VSMC-secreted matrix proteoglycans from apoe^−/− mice had increased LDL binding, which was dramatically reduced when TGF-β was inhibited. VSMCs were treated with vehicle, SAA, or TGF-β for 24 h, then washed and incubated with Alexa-594-labeled LDL for 2 h. SAA- and TGF-β-treated cells had increased LDL binding compared with vehicle treatment. To determine the necessity of TGF-β in SAA-mediated LDL retention, VSMCs were treated with SAA and either the TGF-β-neutralizing antibody 1D11 or control antibody 13C4. The 1D11 prevented the SAA increase in LDL binding; there was no effect of 13C4. To determine the role of proteoglycans in the SAA-mediated LDL binding, cells were treated with sodium chlorate, which prevents sulfation of proteoglycan glycosaminoglycan side chains. Sodium chlorate prevented both the SAA- and TGF-β-dependent increase in LDL binding. LDL binding is expressed as Alexa-fluor 594 surface area normalized to DAPI surface area quantified by fluorescent microscopy using ImageJ software (NIH). Data are presented as mean ± SEM from 5 to 10 20× regions/condition. * P < 0.001.

Fig. 2.

Inhibition of TGF-β prevented ad-SAA increase in vascular biglycan content. The apoe^−/− mice were injected with ad-Null, ad-SAA, or ad-SAA with the TGF-β-neutralizing antibody 1D11 or control antibody 13C4. A: Human SAA and TGF-β were measured in plasma collected 24 h after injections. 1D11 prevented the ad-SAA induction of TGF-β but had no effects on plasma SAA; N = 4–5/group. B: Aortas were collected 28 days after injections and immunoblotted for biglycan (BGN) or actin. The 1D11 prevented the ad-SAA induction of vascular biglycan, but 13C4 had no effect. Each lane shows protein from an individual mouse representative of 4–5 mice per group.

Fig. 3.

Increase in vascular biglycan content after a brief elevation of SAA persists at least 16 weeks. Mice were injected with ad-SAA (black circles), ad-Null (open squares), or saline (black triangles) and fed normal rodent chow for 16 weeks. A: Mice receiving a single injection of ad-SAA had a dramatic albeit transient increase in human SAA. B: All groups had a small, transient increase in murine SAA. C: TGF-β was transiently but dramatically increased in ad-SAA mice compared with ad-Null or saline mice. Shown are means ± SEM from n = 2–6 mice/group per time point. D: After 16 weeks, carotid arteries were collected and immunoblotted for biglycan (BGN) or actin, then analyzed by densitometry using ImageJ software. Each lane shows protein from an individual mouse; densitometry shows means ± SEM for 5–7 mice per group.

Fig. 4.

Atherosclerosis is increased in apoe^−/− mice after only a brief increase in plasma SAA. Mice were injected with ad-SAA, ad-Null, or saline and fed normal rodent chow for 16 weeks. Atherosclerosis was measured at three sites: the aortic intimal surface (A), the brachiocephalic artery (B), and the aortic sinus (C). Atherosclerosis data presented as mean ± SEM; n = 3–20/ group analyzed by one-way ANOVA. D: An atherosclerotic lesion in an aortic root from an ad-SAA-injected apoe^−/− mouse was double stained for apoB (green) and biglycan (red). Colocalization is indicated by yellow. Shown is a confocal image magnified 63×, representative of four mice. The asterisk indicates the lumen of the aortic sinus. Scale bar indicates 10 mm.

Fig. 5.

Atherosclerosis was increased after sustained elevation of human SAA in rag1^−/− × apoe^−/− mice. Mice were injected with ad-SAA (black circles), ad-Null (open squares), or saline (black triangles) every 21 days and fed normal rodent chow for 12 weeks. A: Mice receiving ad-SAA had a significant, persistent elevation of human SAA. B: Murine SAA did not increase nor did it differ between groups throughout the study. C: TGF-β was increased only in mice injected with ad-SAA and remained elevated throughout the study. Shown are means ± SEM from n = 2–6 mice/group per time point. Ad-SAA-injected mice had increased atherosclerosis on the aortic intimal surface (D) and in the brachiocephalic artery (E). There was a trend toward increased atherosclerosis in the aortic root that did not reach significance (F). Data are presented as mean ± SEM; n = 4–16/group analyzed by one-way ANOVA.