Joel C. Thompson

Serum amyloid A in atherosclerosis.

Purpose of review Serum amyloid A (SAA) is a family of acute-phase proteins which are shown to correlate with cardiovascular disease, but whether this SAA contributes causally to atherosclerosis development or reflects underlying disease or risk factors remains unclear. Recent findings SAA has been detected within atherosclerotic lesions and within adipose tissue where it is hypothesized that it may play a contributory role in disease development. In the acute-phase response SAA is synthesized by the liver and transported primarily in association with HDL. However, there is a growing literature suggesting that localized synthesis of SAA within the vasculature, or adipose tissue, may play a distinct role in disease development. Furthermore, SAA can be found in association with apoB-containing lipoproteins, in which its biological activity may be different. Summary This review will discuss recent experimental evidence supporting a causal role of SAA with atherosclerosis.

Angiotensin II increases vascular proteoglycan content preceding and contributing to atherosclerosis development

Angiotensin II (angII) is known to promote atherosclerosis; however, the mechanisms involved are not fully understood. To determine whether angII stimulates proteoglycan production and LDL retention, LDL receptor-deficient mice were infused with angII (1,000 ng/kg/min) or saline via osmotic minipumps. To control for the hypertensive effect of angII, a parallel group received norepinephrine (NE; 5.6 mg/kg/day). Arterial lipid accumulation was evaluated by measuring the retention rate of LDL in isolated carotid arteries perfused ex vivo. Mice infused with angII had increased vascular content of biglycan and perlecan and retained twice as much LDL as saline- or NE-infused mice, although no group developed atherosclerosis at this time. To determine whether this increase in biglycan and perlecan content predisposed to atherosclerosis development, mice were infused with angII, saline, or NE for 4 weeks, then pumps were removed and mice received an atherogenic Western diet for another 6 weeks. Mice that had received angII infusions had 3-fold increased atherosclerosis compared with mice that had received saline or NE, and apolipoprotein B colocalized with both proteoglycans. Thus, one mechanism by which angII promotes atherosclerosis is increased proteoglycan synthesis and increased arterial LDL retention, which precedes and contributes to atherosclerosis development.

Serum Amyloid A, but Not C-Reactive Protein, Stimulates Vascular Proteoglycan Synthesis in a Pro-Atherogenic Manner

Inflammatory markers serum amyloid A (SAA) and C-reactive protein (CRP) are predictive of cardiac disease and are proposed to play causal roles in the development of atherosclerosis, in which the retention of lipoproteins by vascular wall proteoglycans is critical. The purpose of this study was to determine whether SAA and/or CRP alters vascular proteoglycan synthesis and lipoprotein retention in a pro-atherogenic manner. Vascular smooth muscle cells were stimulated with either SAA or CRP (1 to 100 mg/L) and proteoglycans were then isolated and characterized. SAA, but not CRP, increased proteoglycan sulfate incorporation by 50 to 100% in a dose-dependent manner (P < 0.0001), increased glycosaminoglycan chain length, and increased low-density lipoprotein (LDL) binding affinity (K(d), 29 microg/ml LDL versus 90 microg/ml LDL for SAA versus control proteoglycans; P < 0.005). Furthermore, SAA up-regulated biglycan via the induction of endogenous transforming growth factor (TGF)-beta. To determine whether SAA stimulated proteoglycan synthesis in vivo, ApoE(-/-) mice were injected with an adenovirus expressing human SAA-1, a null virus, or saline. Mice that received adenovirus expressing SAA had increased TGF-beta concentrations in plasma and increased aortic biglycan content compared with mice that received either null virus or saline. Thus, SAA alters vascular proteoglycans in a pro-atherogenic manner via the stimulation of TGF-beta and may play a causal role in the development of atherosclerosis.

Renal accumulation of biglycan and lipid retention accelerates diabetic nephropathy.

Hyperlipidemia worsens diabetic nephropathy, although the mechanism by which renal lipids accumulate is unknown. We previously demonstrated that renal proteoglycans have high low-density lipoprotein (LDL) binding affinity, suggesting that proteoglycan-mediated LDL retention may contribute to renal lipid accumulation. The aim of this study was to determine the relative effect of diabetes and hyperlipidemia on renal proteoglycan content. Diabetic and non-diabetic LDL receptor-deficient mice were fed diets containing 0% or 0.12% cholesterol for 26 weeks, and then kidneys were analyzed for renal lipid and proteoglycan content. Diabetic mice on the high-cholesterol diet had accelerated development of diabetic nephropathy with elevations in urine albumin excretion, glomerular and renal hypertrophy, and mesangial matrix expansion. Renal lipid accumulation was significantly increased by consumption of the 0.12% cholesterol diet, diabetes, and especially by both. The renal proteoglycans biglycan and decorin were detectable in glomeruli, with a significant increase in renal biglycan content in diabetic mice on the high-cholesterol diet. Renal biglycan and renal apolipoprotein B were colocalized, and regression analyses showed a significant relation between renal biglycan and renal apolipoprotein B content. The increased renal biglycan content in diabetic nephropathy probably contributes to renal lipid accumulation and the development of diabetic nephropathy.

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

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.

Deficiency of Endogenous Acute Phase Serum Amyloid A Does Not Affect Atherosclerotic Lesions in Apolipoprotein E–Deficient Mice

Objective—Although elevated plasma concentrations of serum amyloid A (SAA) are associated strongly with increased risk for atherosclerotic cardiovascular disease in humans, the role of SAA in the pathogenesis of lesion formation remains obscure. Our goal was to determine the impact of SAA deficiency on atherosclerosis in hypercholesterolemic mice. Approach and Results—Apolipoprotein E–deficient (apoE−/−) mice, either wild type or deficient in both major acute phase SAA isoforms, SAA1.1 and SAA2.1, were fed a normal rodent diet for 50 weeks. Female mice, but not male apoE−/− mice deficient in SAA1.1 and SAA2.1, had a modest increase (22%; P⩽0.05) in plasma cholesterol concentrations and a 53% increase in adipose mass compared with apoE−/− mice expressing SAA1.1 and SAA2.1 that did not affect the plasma cytokine levels or the expression of adipose tissue inflammatory markers. SAA deficiency did not affect lipoprotein cholesterol distributions or plasma triglyceride concentrations in either male or female mice. Atherosclerotic lesion areas measured on the intimal surfaces of the arch, thoracic, and abdominal regions were not significantly different between apoE−/− mice deficient in SAA1.1 and SAA2.1 and apoE−/− mice expressing SAA1.1 and SAA2.1 in either sex. To accelerate lesion formation, mice were fed a Western diet for 12 weeks. SAA deficiency had effect neither on diet-induced alterations in plasma cholesterol, triglyceride, or cytokine concentrations nor on aortic atherosclerotic lesion areas in either male or female mice. In addition, SAA deficiency in male mice had no effect on lesion areas or macrophage accumulation in the aortic roots. Conclusions—The absence of endogenous SAA1.1 and 2.1 does not affect atherosclerotic lipid deposition in apolipoprotein E–deficient mice fed either normal or Western diets.

Increased atherosclerosis in mice with increased vascular biglycan content

OBJECTIVE The response to retention hypothesis of atherogenesis proposes that atherosclerosis is initiated via the retention of atherogenic lipoproteins by vascular proteoglycans. Co-localization studies suggest that of all the vascular proteoglycans, biglycan is the one most closely co-localized with LDL. The goal of this study was to determine if over-expression of biglycan in hyperlipidemic mice would increase atherosclerosis development. METHODS Transgenic mice were developed by expressing biglycan under control of the smooth muscle actin promoter, and were crossed to the LDL receptor deficient (C57BL/6 background) atherosclerotic mouse model. Biglycan transgenic and non-transgenic control mice were fed an atherogenic Western diet for 4-12 weeks. RESULTS LDL receptor deficient mice overexpressing biglycan under control of the smooth muscle alpha actin promoter had increased atherosclerosis development that correlated with vascular biglycan content. CONCLUSION Increased vascular biglycan content predisposes to increased lipid retention and increased atherosclerosis development.

Biglycan deficiency: Increased aortic aneurysm formation and lack of atheroprotection

Proteoglycans of the arterial wall play a critical role in vascular integrity and the development of atherosclerosis owing to their ability to organize extracellular matrix molecules and to bind and retain atherogenic apolipoprotein (apo)-B containing lipoproteins. Prior studies have suggested a role for biglycan in aneurysms and in atherosclerosis. Angiotensin II (angII) infusions into mice have been shown to induce abdominal aortic aneurysm development, increase vascular biglycan content, increase arterial retention of lipoproteins, and accelerate atherosclerosis. The goal of this study was to determine the role of biglycan in angII-induced vascular diseases. Biglycan-deficient or biglycan wildtype mice crossed to LDL receptor deficient (Ldlr-/-) mice (C57BL/6 background) were infused with angII (500 or 1000ng/kg/min) or saline for 28days while fed on normal chow, then pumps were removed, and mice were switched to an atherogenic Western diet for 6weeks. During angII infusions, biglycan-deficient mice developed abdominal aortic aneurysms, unusual descending thoracic aneurysms, and a striking mortality caused by aortic rupture (76% for males and 48% for females at angII 1000ng/kg/min). Histological analyses of non-aneurysmal aortic segments from biglycan-deficient mice revealed a deficiency of dense collagen fibers and the aneurysms demonstrated conspicuous elastin breaks. AngII infusion increased subsequent atherosclerotic lesion development in both biglycan-deficient and biglycan wildtype mice. However, the biglycan genotype did not affect the atherosclerotic lesion area induced by the Western diet after treatment with angII. Biglycan-deficient mice exhibited significantly increased vascular perlecan content compared to biglycan wildtype mice. Analyses of the atherosclerotic lesions demonstrated that vascular perlecan co-localized with apoB, suggesting that increased perlecan compensated for biglycan deficiency in terms of lipoprotein retention. Biglycan deficiency increases aortic aneurysm development and is not protective against the development of atherosclerosis. Biglycan deficiency leads to loosely packed aortic collagen fibers, increased susceptibility of aortic elastin fibers to angII-induced stress, and up-regulation of vascular perlecan content.

Reversibility of renal injury with cholesterol lowering in hyperlipidemic diabetic mice

Hyperlipidemia is a risk factor for development and progression of diabetic nephropathy. However, it is not known if reduction of hyperlipidemia is protective against progression of disease. The goal of this study was to determine if reduction of hypercholesterolemia could limit progression of diabetic nephropathy. Diabetic and nondiabetic LDL receptor deficient (LDLR−/−) mice were fed diets containing either no cholesterol (0%) or high cholesterol (0.12%) for 36 weeks. One group each of diabetic and nondiabetic mice were fed the high-cholesterol diet for 26 weeks then changed to the 0% cholesterol diet for the last 10 weeks. Consumption of the high-cholesterol diet exacerbated the development of diabetic nephropathy with elevations in urine albumin excretion, glomerular and renal hypertrophy, and mesangial matrix expansion. Increased glomerular lipid and apolipoprotein B accumulation was found in diabetic mice that consumed the 0.12% cholesterol diet compared with other groups. However, diabetic mice that changed from the high-cholesterol diet to the 0% cholesterol diet for the last 10 weeks had lower urine albumin excretion and mesangial matrix expansion compared with mice that consumed the 0.12% cholesterol diet throughout. This suggests that hyperlipidemia causes continuous renal injury, and that lowering cholesterol levels by dietary means can improve renal function in diabetic LDLR−/− mice.

Prevention of TGFβ induction attenuates angII-stimulated vascular biglycan and atherosclerosis in Ldlr−/− mice

Angiotensin II (angII) accelerates atherosclerosis, but the mechanisms are not fully understood. The aim of this study was to determine whether TGFβ is required for angII-induced atherosclerosis. Ldlr-null mice fed a normal chow diet were infused with angII or saline for 28 days. A single injection of TGFβ neutralizing antibody 1D11 (2 mg/kg) prevented angII-induction of TGFβ1 levels, and strikingly attenuated angII-induced accumulation of aortic biglycan content. To study atherosclerosis, mice were infused with angII or saline for 4 weeks, and then fed Western diet for a further 6 weeks. 1D11 had no effect on systolic blood pressure or plasma cholesterol; however, angII-infused mice that received 1D11 had reduced atherosclerotic lesion area by 30% (P < 0.05). Immunohistochemical analyses demonstrated that angII induced both lipid retention and accumulation of biglycan and perlecan which colocalized with apoB. 1D11 strikingly reduced the effect of angII on biglycan but not perlecan. 1D11 decreased total collagen content (P < 0.05) in the lesion area without changing plaque inflammation markers (CD68 and CD45). Thus, this study demonstrates that neutralization of TGFβ attenuated angII stimulation of biglycan accumulation and atherogenesis in mice, suggesting that TGFβ-mediated biglycan induction is one of the mechanisms underlying angII-promoted atherosclerosis.