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Background: Obesity increases the risk for human abdominal aortic aneurysms (AAAs) and enhances Ang II (angiotensin II)–induced AAA formation in C57BL/6J mice. Obesity is also associated with increases in perivascular fat that expresses proinflammatory markers including SAA (serum amyloid A). We previously reported that deficiency of SAA significantly reduces Ang II–induced inflammation and AAA in hyperlipidemic apoE-deficient mice. In this study. we investigated whether adipose tissue-derived SAA plays a role in Ang II–induced AAA in obese C57BL/6J mice. Methods: The development of AAA was compared between male C57BL/6J mice (wild type), C57BL/6J mice lacking SAA1.1, SAA2.1, and SAA3 (TKO); and TKO mice harboring a doxycycline-inducible, adipocyte-specific SAA1.1 transgene (TKO-Tg fat ; SAA expressed only in fat). All mice were fed an obesogenic diet and doxycycline to induce SAA transgene expression and infused with Ang II to induce AAA. Results: In response to Ang II infusion, SAA expression was significantly increased in perivascular fat of obese C57BL/6J mice. Maximal luminal diameters of the abdominal aorta were determined by ultrasound before and after Ang II infusion, which indicated a significant increase in aortic luminal diameters in wild type and TKO-TG fat mice but not in TKO mice. Adipocyte-specific SAA expression was associated with MMP (matrix metalloproteinase) activity and macrophage infiltration in abdominal aortas of Ang II–infused obese mice. Conclusions: We demonstrate for the first time that SAA deficiency protects obese C57BL/6J mice from Ang II–induced AAA. SAA expression only in adipocytes is sufficient to cause AAA in obese mice infused with Ang II.

Several studies in the past have reported positive correlations between circulating Serum amyloid A (SAA) levels and obesity. However, based on limited number of studies involving appropriate mouse models, the role of SAA in the development of obesity and obesity-related metabolic consequences has not been established. Accordingly, herein, we have examined the role of SAA in the development of obesity and its associated metabolic complicationsin vivousing mice deficient for all three inducible forms of SAA: SAA1.1, SAA2.1 and SAA3 (TKO). Male and female mice were rendered obese by feeding a high fat, high sucrose diet with added cholesterol (HFHSC) and control mice were fed rodent chow diet. Here, we show that the deletion of SAA does not affect diet-induced obesity, hepatic lipid metabolism or adipose tissue inflammation. However, there was a modest effect on glucose metabolism. The results of this study confirm previous findings that SAA levels are elevated in adipose tissues as well as in the circulation in diet-induced obese mice. However, the three acute phase SAAs do not play a causative role in the development of obesity or obesity-associated adipose tissue inflammation and dyslipidemia.

Background Obesity increases the risk for hypertension in both sexes, but the prevalence of hypertension is lower in females than in males until menopause, despite a higher prevalence of obesity in females. We previously demonstrated that angiotensin-converting enzyme 2 (ACE2), which cleaves the vasoconstrictor, angiotensin II (AngII), to generate the vasodilator, angiotensin-(1-7) (Ang-(1-7)), contributes to sex differences in obesity-hypertension. ACE2 expression in adipose tissue was influenced by obesity in a sex-specific manner, with elevated ACE2 expression in obese female mice. Moreover, estrogen stimulated adipose ACE2 expression and reduced obesity-hypertension in females. In this study, we hypothesized that deficiency of adipocyte ACE2 contributes to obesity-hypertension of females. Methods We generated a mouse model of adipocyte ACE2 deficiency. Male and female mice with adipocyte ACE2 deficiency or littermate controls were fed a low (LF) or a high fat (HF) diet for 16 weeks and blood pressure was quantified by radiotelemetry. HF-fed mice of each sex and genotype were challenged by an acute AngII injection, and blood pressure response was quantified. To translate these findings to humans, we performed a proof-of-principle study in obese transwomen in which systemic angiotensin peptides and blood pressure were quantified prior to and after 12 weeks of gender-affirming 17β-estradiol hormone therapy. Results Adipocyte ACE2 deficiency had no effect on the development of obesity in either sex. HF feeding increased systolic blood pressures (SBP) of wild-type male and female mice compared to LF-fed controls. Adipocyte ACE2 deficiency augmented obesity-induced elevations in SBP in females, but not in males. Obese female, but not obese male mice with adipocyte ACE2 deficiency, had an augmented SBP response to acute AngII challenge. In humans, plasma 17β-estradiol concentrations increased in obese transwomen administered 17β-estradiol and correlated positively with plasma Ang-(1-7)/AngII balance, and negatively to SBP after 12 weeks of 17β-estradiol administration. Conclusions Adipocyte ACE2 protects female mice from obesity-hypertension, and reduces the blood pressure response to systemic AngII. In obese transwomen undergoing gender-affirming hormone therapy, 17β-estradiol administration may regulate blood pressure via the Ang-(1-7)/AngII balance. Electronic supplementary material The online version of this article (10.1186/s13293-019-0260-8) contains supplementary material, which is available to authorized users.

Purpose of review Acute phase serum amyloid A (SAA) is persistently elevated in chronic inflammatory conditions, and elevated levels predict cardiovascular risk in humans. More recently, murine studies have demonstrated that over-expression of SAA increases and deficiency/suppression of SAA attenuates atherosclerosis. Thus, beyond being a biomarker, SAA appears to play a causal role in atherogenesis. The purpose of this review is to summarize the data supporting SAA as a key player in atherosclerosis development. Recent findings A number of pro-inflammatory and pro-atherogenic activities have been ascribed to SAA. However, the literature is conflicted, as recombinant SAA, and/or lipid-free SAA, used in many of the earlier studies, do not reflect the activity of native human or murine SAA, which exists largely lipid-associated. Recent literatures demonstrate that SAA activates the NLRP3 inflammasome, alters vascular function, affects HDL function, and increases thrombosis. Importantly, SAA activity appears to be regulated by its lipid association, and HDL may serve to sequester and limit SAA activity. Summary SAA has many pro-inflammatory and pro-atherogenic activities, is clearly demonstrated to affect atherosclerosis development, and may be a candidate target for clinical trials in cardiovascular diseases.

Objective— SAA (serum amyloid A) is a family of acute-phase reactants that have proinflammatory and proatherogenic activities. SAA is more lipophilic than apoA-I (apolipoprotein A-I), and during an acute-phase response, Approach and Results— Delipidated human SAA was incubated with SAA-free human lipoproteins; then, samples were reisolated by fast protein liquid chromatography, and SAA analyzed by ELISA and immunoblot. Both in vitro and in vivo, we show that SAA associates with any lipoprotein and does not remain in a lipid-free form. Although SAA is preferentially found on high-density lipoprotein, it can exchange between lipoproteins. In the presence of CETP (cholesterol ester transfer protein), there is greater exchange of SAA between lipoproteins. Subjects with diabetes mellitus, but not those with metabolic syndrome, showed altered SAA lipoprotein distribution postprandially. Proteoglycan-mediated lipoprotein retention is thought to be an underlying mechanism for atherosclerosis development. SAA has a proteoglycan-binding domain. Lipoproteins containing SAA had increased proteoglycan binding compared with SAA-free lipoproteins. Conclusions— Thus, SAA is an exchangeable apolipoprotein and increases apoB-containing lipoproteins’ proteoglycan binding. We and others have previously reported the presence of SAA on low-density lipoprotein in individuals with obesity, diabetes mellitus, and metabolic syndrome. We propose that the presence of SAA on apoB-containing lipoproteins may contribute to cardiovascular disease development in these populations.

Vascular biglycan contributes to atherosclerosis development and increased biglycan expression correlates with increased atherosclerosis. However, mice deficient in biglycan have either no reduction in atherosclerosis or an unexpected increase in atherosclerosis. Biglycan deficient mice have systemically elevated TGF-β, likely due to lack of sequestration of TGF-β in the extracellular matrix. The purpose of this study was to determine if prevention of TGF-β elevations in biglycan deficient mice affected atherosclerosis development.Biglycan deficient mice were crossed to Ldlr deficient mice. Diabetes was induced via streptozotocin and all mice were fed a high cholesterol diet. Diabetic biglycan wild type and biglycan deficient Ldlr deficient mice were injected with the TGF-β neutralizing antibody 1D11 or the irrelevant control antibody 13C4.Biglycan deficient mice had significantly elevated plasma TGF-β levels, which was further increased by diabetes, and significantly increased atherosclerosis. There was a significant correlation between TGF-β concentrations and atherosclerosis. However, despite nearly complete suppression of plasma TGF-β levels in mice treated with the TGF-β neutralizing antibody 1D11, there was no significant difference in atherosclerosis between mice with elevated TGF-β levels and mice with suppressed TGF-β levels.The increased atherosclerosis in biglycan deficient mice does not appear to be due to elevations in TGF-β.

Animal studies demonstrate that hyperlipidemia and renal lipid accumulation contribute to the pathogenesis of diabetic nephropathy (DN). We previously demonstrated that renal lipoproteins colocalize with biglycan, a renal proteoglycan. The purpose of this study was to determine whether prevention of renal lipid (apoB) accumulation attenuates DN. Biglycan-deficient and biglycan wild-type Ldlr−/− mice were made diabetic via streptozotocin and fed a high cholesterol diet. As biglycan deficiency is associated with elevated transforming growth factor-β (TGF-β), in some experiments mice were injected with either the TGF-β-neutralizing antibody, 1D11, or with 13C4, an irrelevant control antibody. Biglycan deficiency had no significant effect on renal apoB accumulation, but led to modest attenuation of DN with ∼30% reduction in albuminuria; however, biglycan deficiency caused a striking elevation in TGF-β. Use of 1D11 led to sustained suppression of TGF-β for approximately 8 weeks at a time. The 1D11 treatment caused decreased renal apoB accumulation, decreased albuminuria, decreased renal hypertrophy, and improved survival, compared with the 13C4 treatment. Thus, prevention of renal apoB accumulation is protective against development of DN. Furthermore, this study demonstrates that prevention of renal apoB accumulation is a mechanism by which TGF-β inhibition is nephroprotective.

Disruption of the circadian system caused by disordered exposure to light is pervasive in modern society and increases the risk of cardiovascular disease. The mechanisms by which this happens are largely unknown. ApolipoproteinE-deficient (ApoE−/−) mice are studied commonly to elucidate mechanisms of atherosclerosis. In this study, we determined the effects of light-induced circadian disruption on atherosclerosis in ApoE−/− mice. We first characterized circadian rhythms of behavior, light responsiveness, and molecular timekeeping in tissues from ApoE−/− mice that were indistinguishable from rhythms in ApoE+/+ mice. These data showed that ApoE−/− mice had no inherent circadian disruption and therefore were an appropriate model for our study. We next induced severe disruption of circadian rhythms by exposing ApoE−/− mice to constant light for 12 weeks. Constant light exposure exacerbated atherosclerosis in male, but not female, ApoE−/− mice. Male ApoE−/− mice exposed to constant light had increased serum cholesterol concentrations due to increased VLDL/LDL fractions. Taken together, these data suggest that ApoE−/− mice are an appropriate model for studying light-induced circadian disruption and that exacerbated dyslipidemia may mediate atherosclerotic lesion formation caused by constant light exposure.

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.

Serum amyloid A (SAA) is a family of acute-phase reactants. Plasma levels of human SAA1/SAA2 (mouse SAA1.1/2.1) can increase ≥1,000-fold during an acute-phase response. Mice, but not humans, express a third relatively understudied SAA isoform, SAA3. We investigated whether mouse SAA3 is an HDL-associated acute-phase SAA. Quantitative RT-PCR with isoform-specific primers indicated that SAA3 and SAA1.1/2.1 are induced similarly in livers (∼2,500-fold vs. ∼6,000-fold, respectively) and fat (∼400-fold vs. ∼100-fold, respectively) of lipopolysaccharide (LPS)-injected mice. In situ hybridization demonstrated that all three SAAs are produced by hepatocytes. All three SAA isoforms were detected in plasma of LPS-injected mice, although SAA3 levels were ∼20% of SAA1.1/2.1 levels. Fast protein LC analyses indicated that virtually all of SAA1.1/2.1 eluted with HDL, whereas ∼15% of SAA3 was lipid poor/free. After density gradient ultracentrifugation, isoelectric focusing demonstrated that ∼100% of plasma SAA1.1 was recovered in HDL compared with only ∼50% of SAA2.1 and ∼10% of SAA3. Thus, SAA3 appears to be more loosely associated with HDL, resulting in lipid-poor/free SAA3. We conclude that SAA3 is a major hepatic acute-phase SAA in mice that may produce systemic effects during inflammation.

Background and aims Serum amyloid A (SAA) predicts cardiovascular events. Overexpression of SAA increases atherosclerosis development; however, deficiency of two of the murine acute phase isoforms, SAA1.1 and SAA2.1, has no effect on atherosclerosis. SAA3 is a pseudogene in humans, but is an expressed acute phase isoform in mice. The goal of this study was to determine if SAA3 affects atherosclerosis in mice. Methods ApoE-/- mice were used as the model for all studies. SAA3 was overexpressed by an adeno-associated virus or suppressed using an anti-sense oligonucleotide approach. Results Over-expression of SAA3 led to a 4-fold increase in atherosclerosis lesion area compared to control mice (p = 0.01). Suppression of SAA3 decreased atherosclerosis in mice genetically deficient in SAA1.1 and SAA2.1 (p Conclusions SAA3 augments atherosclerosis in mice. Our results resolve a previous paradox in the literature and support extensive epidemiological data that SAA is pro-atherogenic.

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.

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.

Biglycan (BGN), a small leucine-rich proteoglycan, binds the pro-fibrotic cytokine transforming growth factor β (TGFβ) and inhibits its bioactivity in vitro. Nevertheless, it is controversial whether BGN plays an inhibitory role in vivo. Therefore, the purpose of this study was to evaluate the effect of BGN deficiency on TGFβ activity in vivo by studying 1-year-old Bgn null and wild-type (WT) mice on an Ldlr-null background. Phenotypic and metabolic characterization showed that the Bgn null mice had lower body weight, shorter body length, and shorter femur length (all p < 0.05). Surprisingly, the Bgn null mice also exhibited a striking reduction in percent body fat compared to WT mice (p == 0.006), but no changes were observed in plasma triglycerides, total cholesterol, or glycohemoglobin. Both total and bioactive TGFβ1 concentrations in plasma were markedly elevated in Bgn null mice compared to WT mice (4-fold and 11-fold increase, respectively, both p < 0.001), but no changes were found in hepatic levels of mRNA for Tgfβ1 or its receptors. Bgn null mice exhibited elevated expression of hepatic fibronectin protein (p = 0.034) without changes in hepatic or renal histology, and Bgn null mice had decreased urinary albumin/creatinine ratio (p = 0.01). Two key downstream targets of bone morphogenetic protein 4-like signaling, SMAD1/3/5 phosphorylation and Id2 gene expression, were found dramatically reduced in Bgn null livers (p = 0.034). Thus, BGN deficiency decreases body fat in this hyperlipidemic mouse model without changing liver or kidney histology. Overall, we propose that this unexpected phenotype arises from the effects of BGN deficiency in vivo to elevate TGFβ levels while decreasing bone morphogenetic protein 4-like signaling.

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.

The ‘response to retention’ hypothesis of atherogenesis proposes that proteoglycans bind and retain low-density lipoproteins (LDL) in the vessel wall. Platelet-derived growth factor (PDGF) is strongly implicated in atherosclerosis and stimulates proteoglycan synthesis. Here we investigated the action of the PDGF receptor inhibitor imatinib on PDGF-mediated proteoglycan biosynthesis in vitro, lipid deposition in the aortic wall in vivo and the carotid artery ex vivo. In human vSMCs, imatinib inhibited PDGF mediated 35S-SO4 incorporation into proteoglycans by 31% (P < 0.01) and inhibited PDGF-mediated size increases in both chemically cleaved and xyloside associated glycosaminoglycan (GAG) chains by 19%, P < 0.05 and 27%, P < 0.05, respectively. Imatinib decreased PDGF stimulation of the 6:4 position sulphation ratio of disaccharides. The half maximal saturation value for LDL binding for proteoglycans from PDGF stimulated cells in the presence of imatinib was approximately 2.5-fold higher than for PDGF treatment alone. In high fat fed ApoE−/– mice, imatinib reduced total lipid staining area by ∼31% (P < 0.05). Carotid artery lipid accumulation in imatinib treated mice was also reduced. Furthermore, we demonstrate that imatinib inhibits phosphorylation of tyrosine 857, the autophosphorylation site of the PDGF receptor, in vSMCs. Thus imatinib inhibits GAG synthesis on vascular proteoglycans and reduces LDL binding in vitro and in vivo and this effect is mediated via the PDGF receptor. These findings validate a novel mechanism to prevent cardiac disease.

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.

Septic shock results from an uncontrolled inflammatory response, mediated primarily by LPS. Cholesterol transport plays an important role in the host response to LPS, as LPS is neutralized by lipoproteins and adrenal cholesterol uptake is required for antiinflammatory glucocorticoid synthesis. In this study, we show that scavenger receptor B-I (SR-BI), an HDL receptor that mediates HDL cholesterol ester uptake into cells, is required for the normal antiinflammatory response to LPS-induced endotoxic shock. Despite elevated plasma HDL levels, SR-BI-null mice displayed an uncontrollable inflammatory cytokine response and a markedly higher lethality rate than control mice in response to LPS. In addition, SR-BI-null mice showed a lack of inducible glucocorticoid synthesis in response to LPS, bacterial infection, stress, or ACTH. Glucocorticoid insufficiency in SR-BI-null mice was due to primary adrenal malfunction resulting from deficient cholesterol delivery from HDL. Furthermore, corticosterone supplementation decreased the sensitivity of SR-BI-null mice to LPS. Plasma from control and SR-BI-null mice exhibited a similar ability to neutralize LPS, whereas SR-BI-null mice showed decreased plasma clearance of LPS into the liver and hepatocytes compared with normal mice. We conclude that SR-BI in mice is required for the antiinflammatory response to LPS-induced endotoxic shock, likely through its essential role in facilitating glucocorticoid production and LPS hepatic clearance.

Background Diabetes is associated with accelerated atherosclerosis, but the mechanisms responsible for this are not known. Proteoglycans have been shown to play a critical role in the initiation of atherosclerosis owing to their ability to bind and retain atherogenic lipoproteins in the artery wall. Proteoglycan structure and composition are altered in atherosclerotic lesions compared with adjacent normal regions of the artery wall, and this is exaggerated in diabetes. The purpose of this study was to determine if metabolic factors associated with diabetes lead to altered proteoglycan structure and composition. Methods Vascular smooth muscle cells, endothelial cells, and macrophages were exposed to normal (5.6 mmol/L) or high (25 mmol/L) glucose levels, various insulin and free fatty acid levels, and the cytokines transforming growth factor β (TGF-β1) and platelet-derived growth factor, alone or in combination, and proteoglycan synthesis was determined. Results Glucose concentrations, insulin, and free fatty acids did not alter proteoglycan synthesis, size, or relative distribution. The effect of TGF-β to increase biglycan and versican synthesis, increase sulfate incorporation, and increase the size of the secreted proteoglycans was not altered by the ambient glucose level in the culture medium, nor did high glucose increase levels of active TGF-β. Conclusion Vascular proteoglycan synthesis is not affected by metabolic factors associated with diabetes. We suggest that elevated TGF-β levels in diabetes are responsible for the altered proteoglycan synthesis observed in diabetes.

Objective— Thrombin signaling promotes atherosclerosis by initiating inflammatory events indirectly through platelet activation and directly via protease-activated receptors. Therefore, endogenous thrombin inhibitors may be relevant modulators of atheroprogression and cardiovascular risk. In addition, endogenous thrombin inhibitors may affect the response to non–vitamin K-dependent oral anticoagulants. Here, the question was addressed whether the small leucine-rich proteoglycan biglycan acts as an endogenous thrombin inhibitor in atherosclerosis through activation of heparin cofactor II. Approach and Results— Biglycan concentrations were elevated in the plasma of patients with acute coronary syndrome and in male Apolipoprotein E -deficient ( ApoE −/− ) mice. Biglycan was detected in the glycocalyx of capillaries and the subendothelial matrix of arterioles of ApoE −/− mice and in atherosclerotic plaques. Thereby a vascular compartment is provided that may mediate the endothelial and subendothelial activation of heparin cofactor II through biglycan. ApoE and Bgn double-deficient ( ApoE −/− /Bgn −/0 ) mice showed higher activity of circulating thrombin, increased platelet activation and platelet adhesion in vivo, supporting a role of biglycan in balancing thrombin activity. Furthermore, concentrations of circulating cytokines and aortic macrophage content were elevated in ApoE −/− /Bgn −/0 mice, suggesting a proinflammatory phenotype. Elevated platelet activation and macrophage accumulation were reversed by treating ApoE −/− /Bgn −/0 mice with the thrombin inhibitor argatroban. Ultimately, ApoE −/− /Bgn −/0 mice developed aggravated atherosclerosis. Conclusions— The present results indicate that biglycan plays a previously unappreciated protective role during the progression of atherosclerosis by inhibiting thrombin activity, platelet activation, and finally macrophage-mediated plaque inflammation.

The role of proteoglycans in atherosclerosis has been under increasing study lately. Proteoglycans are a family of molecules composed of a core protein with attached glycosaminoglycan chains. As a class they are ubiquitous, although different proteoglycan species have different tissue distributions and expression patterns. In the vasculature, extracellular matrix proteoglycans, especially those in the small leucine rich repeat class, have several putative roles in atherosclerosis. Proteoglycans are thought to have a role in collagen fibrillogenesis and the organization and structure of the extracellular matrix. As such, changes in the proteoglycan composition of the matrix can affect matrix stability, elasticity, tensile strength, and other functions1. In addition to their roles in extracellular matrix organization, a number of proteoglycans have been shown to have a role in the regulation of cytokines and growth factors including TGF-β2. Thus, changes in the proteoglycan composition of the vasculature may alter the bioavailability of signaling molecules that can have pathogenic consequences. As an example, overexpression of decorin via an adenoviral vector in apoE−/− mice was shown to decrease the progression of atherosclerosis, and the authors suggested that this may be due to the reduction in circulating free TGF-β observed3. Recently, additional studies have described a role for soluble proteoglycans in the regulation of inflammation. For example biglycan, primarily in its soluble form released from matrix during tissue injury, has been shown to interact with a number of molecules including bone morphogenic proteins (BMP)-2,4,6, TGF-β, TNF-α, VEGF, and is a ligand for a number of receptors including the toll-like receptors (TLR)-2 and 4 (for review see 4). Other putative roles for proteoglycans in the vasculature include the regulation of vascular smooth muscle proliferation and migration5,6. Furthermore, as outlined in the “response to retention hypothesis” proteoglycan-mediated lipid retention is thought to be one of the initiating steps in atherosclerosis development7. Positively charged motifs on apolipoproteins B and E can ionically interact with negatively charged sulfate and carboxylic acid groups on glycosaminoglycans, leading to prolonged retention of atherogenic lipoproteins in the subendothelial space. Co-localization studies have suggested that in humans biglycan is a key proteoglycan mediating lipid retention8,9, whereas in mice both biglycan and perlecan co-localize with apolipoproteins10,11. However, the role of biglycan in atherosclerosis development is unclear: we recently demonstrated that overexpression of biglycan increased atherosclerosis, but biglycan deficiency was not protective12,13. In these studies we demonstrated increased vascular perlecan content in biglycan deficient mice suggesting a compensatory response of the vasculature for the biglycan deficiency12. However, the role of perlecan in atherosclerosis is also unclear: decreased vascular perlecan content (using a heterozygous model as the perlecan deficient mouse is not viable) was shown to have decreased early atherosclerosis, but not later atherosclerosis in the apoE−/− model, and no effect in the LDL receptor deficient model14. Thus, various proteoglycans appear to play a variety of roles in atherosclerosis development, but their effects vary and definitive proof of a critical role for proteoglycans remains elusive.

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.

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.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.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.Increased vascular biglycan content predisposes to increased lipid retention and increased atherosclerosis development.

Despite numerous clinical and animal studies, the role of sex steroid hormones on lipoprotein metabolism and atherosclerosis remain controversial.We sought to determine the effects of endogenous estrogen and testosterone on lipoprotein levels and atherosclerosis using mice fed a low-fat diet with no added cholesterol.Male and female low-density lipoprotein receptor-deficient mice were fed an open stock low-fat diet (10% of kcals from fat) for 2, 4, or 17 weeks. Ovariectomy, orchidectomy, or sham surgeries were performed to evaluate the effects of the presence or absence of endogenous hormones on lipid levels, lipoprotein distribution, and atherosclerosis development.Female mice fed the study diet for 17 weeks had a marked increase in levels of total cholesterol, triglycerides, apolipoprotein-B containing lipoproteins, and atherosclerosis compared with male mice. Surprisingly, ovariectomy in female mice had no effect on any of these parameters. In contrast, castration of male mice markedly increased total cholesterol concentrations, triglycerides, apolipoprotein B-containing lipoproteins, and atherosclerotic lesion formation compared with male and female mice.These data suggest that endogenous androgens protect against diet-induced increases in cholesterol concentrations, formation of proatherogenic lipoproteins, and atherosclerotic lesions formation. Conversely orchidectomy, which decreases androgen concentrations, promotes increases in cholesterol concentrations, proatherogenic lipoprotein formation, and atherosclerotic lesion formation in low-density lipoprotein receptor-deficient mice in response to a low-fat diet.

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.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.This review will discuss recent experimental evidence supporting a causal role of SAA with atherosclerosis.

Atherosclerosis is accelerated in the setting of diabetes, but the factors driving this phenomenon remain elusive. Hyperglycemia leads to elevated levels of transforming growth factor (TGF)-β and TGF-β has been implicated as a factor in atherosclerosis. Given the established association between hyperglycemia and elevated TGF-β, it is plausible that elevated TGF-β levels in diabetes play a pathogenic role in the development of accelerated atherosclerosis. TGF-β is a potent regulator of extracellular matrix synthesis, including many actions on proteoglycan synthesis that lead to increased binding to low-density lipoprotein and therefore potentially increased lipid retention in the vessel wall and accelerated atherosclerosis. TGF-β signals through the canonical TGF-β receptor I-mediated phosphorylation of Smad transcription factors and TGF-β signaling is also known to involve, positively and negatively, interactions with the mitogen-activated protein kinase pathways. The focus of the present review is on the effects of TGF-β on proteoglycan synthesis in vascular smooth muscle and particularly the signaling pathways through which TGF-β exerts its effects, because those pathways may be therapeutic targets for the prevention of pathological modifications in the proteoglycan component of the vessel wall in the vascular diseases of diabetes.

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.

Background— Strengthening the macrophage glutathione redox buffer reduces macrophage content and decreases the severity of atherosclerotic lesions in LDL receptor-deficient (LDLR −/− ) mice, but the underlying mechanisms were not clear. This study examined the effect of metabolic stress on the thiol redox state, chemotactic activity in vivo, and the recruitment of macrophages into atherosclerotic lesions and kidneys of LDL-R −/− mice in response to mild, moderate, and severe metabolic stress. Methods and Results— Reduced glutathione (GSH) and glutathione disulfide (GSSG) levels in peritoneal macrophages isolated from mildly, moderately, and severe metabolically-stressed LDL-R −/− mice were measured by HPLC, and the glutathione reduction potential (E h ) was calculated. Macrophage E h correlated with the macrophage content in both atherosclerotic (r 2 =0.346, P =0.004) and renal lesions (r 2 =0.480, P =0.001) in these mice as well as the extent of both atherosclerosis (r 2 =0.414, P =0.001) and kidney injury (r 2 =0.480, P =0.001). Compared to LDL-R −/− mice exposed to mild metabolic stress, macrophage recruitment into MCP-1-loaded Matrigel plugs injected into LDL-R −/− mice increased 2.6-fold in moderately metabolically-stressed mice and 9.8-fold in severely metabolically-stressed mice. The macrophage E h was a strong predictor of macrophage chemotaxis (r 2 =0.554, P Conclusion— Thiol oxidative stress enhances macrophage recruitment into vascular and renal lesions by increasing the responsiveness of macrophages to chemoattractants. This novel mechanism contributes at least in part to accelerated atherosclerosis and kidney injury associated with dyslipidemia and diabetes in mice.

The epidemic of obesity sweeping developed nations is accompanied by an increase in atherosclerotic cardiovascular diseases. Dyslipidemia, diabetes, hypertension, and obesity are risk factors for cardiovascular disease. However, delineating the mechanism of obesity-accelerated atherosclerosis has been hampered by a paucity of animal models. Similar to humans, apolipoprotein E-deficient (apoE(-/-)) mice spontaneously develop atherosclerosis over their lifetime. To determine whether apoE(-/-) mice would develop obesity with accelerated atherosclerosis, we fed mice diets containing 10 (low fat (LF)) or 60 (high fat (HF)) kcal % from fat for 17 weeks. Mice fed the HF diet had a marked increase in body weight and atherosclerotic lesion formation compared to mice fed the LF diet. There were no significant differences between groups in serum total cholesterol, triglycerides, or leptin concentrations. Plasma concentrations of the acute-phase reactant serum amyloid A (SAA) are elevated in both obesity and cardiovascular disease. Accordingly, plasma SAA concentrations were increased fourfold (P0.01) in mice fed the HF diet. SAA was associated with both pro- and antiatherogenic lipoproteins in mice fed the HF diet compared to those fed the LF diet, in which SAA was primarily associated with the antiatherogenic lipoprotein high-density lipoprotein (HDL). Moreover, SAA was localized with apoB-containing lipoproteins and biglycan in the vascular wall. Taken together, these data suggest male apoE-deficient mice are a model of metabolic syndrome and that chronic low level inflammation associated with increased SAA concentrations may mediate atherosclerotic lesion formation.

The response to retention hypothesis outlines the initial stages of atherosclerotic lesion formation. The central theme of the hypothesis is that proteoglycan mediated lipoprotein retention plays a critical step in the initiation of atherosclerosis development. Recent research using human arterial specimens, transgenic mouse models and molecular biology techniques have added to our understanding of atherosclerosis development, and provided experimental data in support of the response to retention hypothesis. In this review we summarize the recent data, in particular that which addresses mechanisms by which diabetes can accelerate atherosclerosis formation, with a focus on proteoglycan-mediated LDL retention.

Glucosamine, commonly consumed for the treatment of osteoarthritis, is classified as a nutritional supplement; however, there are few data regarding its metabolic or vascular effects. Glucosamine is a component of the hexosamine pathway, which has been implicated in the development of insulin resistance. Anecdotal reports suggest that glucosamine consumption can increase circulating cholesterol concentrations. To investigate the metabolic and vascular effects of glucosamine supplementation, we studied male and female LDL receptor-deficient mice fed a Western diet (21% fat, 0.15% cholesterol). Three groups of 6-10 mice of each gender received either no supplement, 15 mg . kg(-1) . d(-1) glucosamine (equivalent to an average human dose), or 50 mg . kg(-1) . d(-1) glucosamine added to their drinking water for 5, 10, or 20 wk. Plasma cholesterol and triglyceride concentrations increased in all mice with the addition of the Western diet. However, after 20 wk of treatment, cholesterol and triglyceride concentrations increased further in male mice consuming glucosamine compared with control groups. Glucosamine-supplemented mice had increased initiation of atherosclerosis after 5 wk; however, there was no effect on progression of atherosclerosis in either gender after longer periods of glucosamine supplementation (10 or 20 wk). Although long-term glucosamine supplementation exacerbated the hyperlipidemia in male mice, no increase in atherosclerosis occurred. Thus, glucosamine supplementation appears to be safe, with no adverse vascular consequences.

The retention of atherogenic lipoproteins in the artery wall through their interactions with the arterial extracellular matrix is a critical step in the development of atherosclerosis, as outlined in the 'response to retention' hypothesis. Lipoprotein retention by vascular proteoglycans is thought to be the principle means of lipoprotein retention, although lipoprotein binding to other components of the extracellular matrix has been reported. The interactions of lipoproteins and proteoglycans can be direct through ionic interactions between the negatively charged glycosaminoglycan chains of proteoglycans and positively charged residues of apolipoproteins B and E, or can be mediated through bridging molecules such as lipoprotein lipase. Retention of atherogenic lipoproteins within the artery wall environment leads to pathophysiologically important modifications of the lipoproteins, including oxidation. Oxidation of lipoproteins leads to increased uptake by macrophages, leading to the formation of foam cells. This article reviews the scientific evidence in support of the response to retention hypothesis, with a specific focus on the effects of diabetes to modify lipoprotein retention.

Retention of atherogenic lipoproteins in the artery wall by proteoglycans is a key step in the development of atherosclerosis. Thiazolidinediones have been shown to reduce atherosclerosis in mouse models. The aim of this study was to determine whether thiazolidinediones modify vascular proteoglycan synthesis in a way that decreases LDL binding.Primate aortic smooth muscle cells were exposed to troglitazone or rosiglitazone, or no stimulus at all for a 24-hour steady-state labelling period. Sulphate incorporation, size and LDL binding affinity of proteoglycans were determined. Proteoglycans secreted by cells in the presence or absence of troglitazone were separated into large and small classes by size exclusion chromatography, and LDL binding affinity was determined.Proteoglycans synthesised by cells exposed to troglitazone or rosiglitazone were smaller, with decreased sulphate incorporation and decreased LDL binding affinity. However, troglitazone had a greater effect than rosiglitazone. Troglitazone reduced the LDL binding affinities of both the large and small proteoglycans compared with control. The binding differences persisted when glycosaminoglycan chains released from proteoglycans were incubated with LDL, indicating that troglitazone affects the glycosaminoglycan synthetic machinery of these cells.Thiazolidinediones decrease the LDL binding affinity of the proteoglycans synthesised by primate aortic smooth muscle cells. This could, in part, account for the reduced atherosclerosis observed in animal models.

Retention of LDL in the artery intima is mediated by extracellular matrix proteoglycans and plays an important role in the initiation of atherosclerosis. Compared with quiescent cells, proliferating smooth muscle cells secrete proteoglycans with elongated glycosaminoglycan side chains, which have an increased binding affinity to LDL. Because 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) decrease smooth muscle cell proliferation, we hypothesized that statin exposure would decrease both the size and LDL binding affinity of vascular proteoglycans. Monkey aortic smooth muscle cells grown in culture were exposed to simvastatin (10 and 100 microM) and cerivastatin (0.1 and 1 microM), and newly secreted proteoglycans were quantified and characterized. Both simvastatin and cerivastatin caused a concentration-dependent reduction in cell growth and reduced 35SO4 incorporation into secreted proteoglycans, on both an absolute and a per cell basis. Interestingly, statin exposure increased the apparent molecular weight and hydrodynamic size of secreted proteoglycans. However, proteoglycans secreted from statin-exposed cells demonstrated a reduction in binding affinity to LDL. Thus, statins may induce atheroprotective changes in vascular proteoglycans and lower LDL retention in the vessel wall. These findings suggest a mechanism whereby statins may benefit atherosclerosis in a manner unrelated to serum LDL lowering.

Diabetes in humans accelerates cardiovascular disease caused by atherosclerosis. The relative contributions of hyperglycemia and dyslipidemia to atherosclerosis in patients with diabetes are not clear, largely because there is a lack of suitable animal models. We therefore have developed a transgenic mouse model that closely mimics atherosclerosis in humans with type 1 diabetes by breeding low-density lipoprotein receptor-deficient mice with transgenic mice in which type 1 diabetes can be induced at will. These mice express a viral protein under control of the insulin promoter and, when infected by the virus, develop an autoimmune attack on the insulin-producing beta cells and subsequently develop type 1 diabetes. When these mice are fed a cholesterol-free diet, diabetes, in the absence of associated lipid abnormalities, causes both accelerated lesion initiation and increased arterial macrophage accumulation. When diabetic mice are fed cholesterol-rich diets, on the other hand, they develop severe hypertriglyceridemia and advanced lesions, characterized by extensive intralesional hemorrhage. This progression to advanced lesions is largely dependent on diabetes-induced dyslipidemia, because hyperlipidemic diabetic and nondiabetic mice with similar plasma cholesterol levels show a similar extent of atherosclerosis. Thus, diabetes and diabetes-associated lipid abnormalities have distinct effects on initiation and progression of atherosclerotic lesions.

Atherosclerosis is the main cause of morbidity and mortality in diabetes, yet the underlying mechanisms remain unclear. Retention of atherogenic lipoproteins by vascular proteoglycans is thought to play a key role in the development of atherosclerotic lesions. High glucose levels cause a variety of diabetic complications by several mechanisms, including upregulation of the hexosamine pathway. Glucosamine, a component of the hexosamine pathway, is a precursor for the synthesis of glycosaminoglycan components of proteoglycans. This study evaluated whether high glucose or glucosamine supplementation of vascular smooth muscle cells would increase proteoglycan synthesis, leading to increased lipoprotein retention. Aortic smooth muscle cells were exposed to physiologic (5.6 mM) or high (25 mM) glucose levels, such as seen in diabetes, or to glucosamine (12 mM). Extracellular proteoglycans were characterized by sulfate incorporation, molecular sieve chromatography, and SDS-PAGE. LDL interactions were assessed by affinity chromatography and gel mobility shift assay. Proteoglycans synthesized in the presence of high glucose demonstrated no differences in size, sulfate incorporation, or LDL binding affinity compared with proteoglycans synthesized under physiological glucose conditions. However, proteoglycans synthesized in the presence of glucosamine had smaller glycosaminoglycan chains than control proteoglycans with a corresponding decrease in lipoprotein retention.Thus, glucose and glucosamine have different effects on proteoglycan biosynthesis and different effects on lipoprotein retention.

Despite the complexities and intricacies of human atheroscleosis our understanding of the underlying processes is growing, in o small part due to the use of animal models of atherosclerosis. uman atherosclerosis is difficult to study as the lesions evolve ver years to decades. However, by utilizing small animal models nwhich atherogenesis can be induced over shorter periods of time weeks tomonths) current research can investigate the pathogenic echanisms involved at various stages of development. Much of ur recent advances in atherosclerosis research have come from he use of murine models, in which atherosclerosis can be induced ia genetic and/or dietarymanipulations over relatively short perids of time. Further, the ability to fairly easily alter the mouse enome, for example over-expressing or deleting a gene of interest, as allowed direct testing of the role of certain proteins in varius stages of atherosclerosis development. The relative low cost, asy access, high fertility, and availability of numerous biological ools havemademurinemodels the apparent first choice for animal tudiesof atherosclerosis.However, therearenumerousdrawbacks o the use of murine models that are often overlooked, and new odels of atherosclerosis are urgently neededby the research comunity to expand our understanding and allow research beyond he limitations of murine models. Although murine models have many features making them dvantageous for use in atherosclerosis studies, there are several ajor concerns. First, mice carry their cholesterol primarily in HDL articles, unless genetically altered. Second, mice do not express holesteryl ester transfer protein (CETP), which plays a signifiant role in lipoprotein metabolism in humans [1]. Third, murine therosclerosis is limited to early stages of disease andmostmodels o not develop advanced atheroma, with none manifesting plaque upture. Other rodent models, including guinea pigs and hamsters