Atherosclerosis-susceptible Mice Research Articles

Recombinant human proteoglycan 4 lowers inflammation and atherosclerosis susceptibility in female low-density lipoprotein receptor knockout mice.

Recombinant human proteoglycan 4 (rhPRG4) is a macromolecular mucin-like glycoprotein that is classically studied as a lubricant within eyes and joints. Given that endogenously produced PRG4 is present within atherosclerotic lesions and genetic PRG4 deficiency increases atherosclerosis susceptibility in mice, in the current study we investigated the anti-atherogenic potential of chronic rhPRG4 treatment. Female low-density lipoprotein receptor knockout mice were fed an atherogenic Western-type diet for 6 weeks and injected three times per week intraperitoneally with 0.5mg rhPRG4 or PBS as control. Treatment with rhPRG4 was associated with a small decrease in plasma-free cholesterol levels, without a change in cholesteryl ester levels. A marked increase in the number of peritoneal foam cells was detected in response to the peritoneal rhPRG4 administration, which could be attributed to elevated peritoneal leukocyte MSR1 expression levels. However, rhPRG4-treated mice exhibited significantly smaller aortic root lesions of 278 ± 21 × 103μm2 compared with 339 ± 15 × 103μm2 in the aortic root of control mice. The overall decreased atherosclerosis susceptibility coincided with a shift in the monocyte and macrophage polarization states towards the patrolling and anti-inflammatory M2-like phenotypes, respectively. Furthermore, rhPRG4 treatment significantly reduced macrophage gene expression levels as well as plasma protein levels of the pro-inflammatory/pro-atherogenic cytokine TNF-alpha. In conclusion, we have shown that peritoneal administration and subsequent systemic exposure to rhPRG4 beneficially impacts the inflammatory state and reduces atherosclerosis susceptibility in mice. Our findings highlight that PRG4 is not only a lubricant but also acts as an anti-inflammatory agent. KEY POINTS: Endogenously produced proteoglycan 4 is found in atherosclerotic lesions and its genetic deficiency in mice is associated with enhanced atherosclerosis susceptibility. In this study we investigated the anti-atherogenic potential of chronic treatment with recombinant human PRG4 in hypercholesterolaemic female low-density lipoprotein receptor knockout mice. We show that recombinant human PRG4 stimulates macrophage foam cell formation, but also dampens the pro-inflammatory state of monocyte/macrophages, eventually leading to a significant reduction in plasma TNF-alpha levels and a lowered atherosclerosis susceptibility. Our findings highlight that peritoneal recombinant human PRG4 treatment can execute effects both locally and systemically and suggest that it will be of interest to study whether rhPRG4 treatment is also able to inhibit the progression and/or induce regression of previously established atherosclerotic lesions.

Metabolic and Cardiovascular Impact of Ketotic Extreme Carbohydrate Restriction

Human trials of ketogenic diets (KDs) have shown promise in weight and glucose management in subjects with type 2 diabetes (T2D), prompting organizations, including Diabetes Canada, to condone their use through recent position statements. However, the effects of intervention with strict dietary regimes on the progression of cardiovascular disease and glucoregulatory processes, like the incretin effect, have not been fully elucidated. The incretins, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), potentiate insulin secretion postprandially to control glycemia but are rapidly inactivated by dipeptidyl peptidase-4 (DPP4) cleavage. In this study, we used atherosclerosis-susceptible mice with diet-induced obesity and dysglycemia to investigate the effect of intervention with extreme carbohydrate restriction. PCSK9 overexpressing mice were fed a high fat, high cholesterol diet (HFD) for 10 weeks, followed by a 12-week continuation or intervention with a KD or chow diet. Mice fed a KD diet had elevated free fatty acids and ketone bodies in the fed state and lower circulating DPP4. Elevated GIP and hyperinsulinemia were also reversed. KD mice had increased insulin secretion in response to 10 mM glucose supplemented with GLP-1 and GIP than HFD mice during ex-vivo perifusion analysis. Additionally, liver triglycerides, cholesterol, and aortic sinus atherosclerosis significantly improved with ketogenic and chow interventions. Nanostring analysis of the aorta demonstrated differentially expressed inflammatory genes and pathways in KD versus HFD feeding. Understanding how dietary regimens drive the metabolic milieu and surrounding pathologies will advance personalized nutrition advice for glycemic control.

Bone Marrow Ts65Dn Trisomy-Induced Changes in Platelet Functionality and Lymphocytopenia Do Not Impact Atherosclerosis Susceptibility in Mice.

The genetic disorder Down syndrome is associated with a decreased susceptibility for atherosclerotic cardiovascular disease. Hematological and immune abnormalities occur frequently in Down syndrome patients. We evaluated, in a preclinical setting, the impact of a Down syndrome-like hematological/immune phenotype on atherosclerosis susceptibility. Hereto, hypercholesterolemic low-density lipoprotein receptor knockout mice were transplanted with bone marrow from either a trisomic Ts65Dn mouse or euploid wild-type control and subsequently fed a Western-type diet to induce the development of atherosclerotic lesions. T and B cell concentrations were markedly reduced in blood of Ts65Dn bone marrow recipients (p < 0.001). Expression levels of the pro-atherogenic scavenger receptor CD36 were respectively 37% and 59% lower (p < 0.001) in trisomic monocytes and macrophages. However, these combined effects did not translate into an altered atherosclerosis susceptibility. Notably, blood platelet numbers were elevated in Ts65Dn bone marrow recipients (+57%; p < 0.001), which was paralleled by higher platelet GPVI protein expression (+35%; p < 0.001) and an enhanced collagen-induced platelet activation (p < 0.001). In conclusion, we have shown that providing mice with a Down syndrome-like hematological profile does not change the susceptibility to atherosclerosis. Furthermore, our studies have uncovered a novel effect of the trisomy on platelet functionality that may be relevant in human clinical settings.

Hematopoietic upstream stimulating factor 1 deficiency is associated with increased atherosclerosis susceptibility in LDL receptor knockout mice

Total body upstream stimulatory factor 1 (USF1) deficiency in mice is associated with brown adipose tissue activation and a marked protection against the development of obesity and atherosclerotic lesions. Functional expression of USF1 has also been detected in monocytes and monocyte-derived macrophages. In the current study we therefore tested whether selective hematopoietic USF1 deficiency can also beneficially impact the development of atherosclerosis. For this purpose, LDL receptor knockout mice were transplanted with bone marrow from USF1 knockout mice or their wild-type littermate controls and subsequently fed a Western-type diet for 20 weeks to stimulate atherosclerotic lesion development. Strikingly, absence of USF1 function in bone marrow-derived cells was associated with exacerbated blood leukocyte (+ 100%; P < 0.01) and peritoneal leukocyte (+ 50%; P < 0.05) lipid loading and an increased atherosclerosis susceptibility (+ 31%; P < 0.05). These effects could be attributed to aggravated hyperlipidemia, i.e. higher plasma free cholesterol (+ 33%; P < 0.001) and cholesteryl esters (+ 39%; P < 0.001), and the development of hepatosteatosis. In conclusion, we have shown that hematopoietic USF1 deficiency is associated with an increased atherosclerosis susceptibility in LDL receptor knockout mice. These findings argue against a contribution of macrophage-specific USF1 deficiency to the previously described beneficial effect of total body USF1 deficiency on atherosclerosis susceptibility in mice.

CLEC-2-dependent platelet subendothelial accumulation by flow disturbance contributes to atherogenesis in mice

Rationale: Platelets play an essential role in atherosclerosis, but the underlying mechanisms remain to be addressed. This study is to investigate the role of platelets in d-flow induced vascular inflammation and the underlying mechanism.Methods: We established a disturbed blood flow (d-flow) model by partial carotid ligation (PCL) surgery using atherosclerosis-susceptible mice and wild-type mice to observe the d-flow induced platelet accumulation in the subendothelium or in the plaque by immunostaining or transmission electron microscopy. The mechanism of platelet subendothelial accumulation was further explored by specific gene knockout mice.Results: We observed presence of platelets in atherosclerotic plaques either in the atheroprone area of aortic arch or in carotid artery with d-flow using Ldlr-/- or ApoE-/- mice on high fat diet. Immunostaining showed the subendothelial accumulation of circulating platelets by d-flow in vivo. Transmission electron microscopy demonstrated the accumulation of platelets associated with monocytes in the subendothelial spaces. The subendothelial accumulation of platelet-monocyte/macrophage aggregates reached peak values at 2 days after PCL. In examining the molecules that may mediate the platelet entry, we found that deletion of platelet C-type lectin-like receptor 2 (CLEC-2) reduced the subendothelial accumulation of platelets and monocytes/macrophages by d-flow, and ameliorated plaque formation in Ldlr-/- mice on high fat diet. Supportively, CLEC-2 deficient platelets diminished their promoting effect on the migration of mouse monocyte/macrophage cell line RAW264.7. Moreover, monocyte podoplanin (PDPN), the only ligand of CLEC-2, was upregulated by d-flow, and the myeloid-specific PDPN deletion mitigated the subendothelial accumulation of platelets and monocytes/macrophages.Conclusions: Our results reveal a new CLEC-2-dependent platelet subendothelial accumulation in response to d-flow to regulate vascular inflammation.

Disruption of Phospholipid Transfer Protein-Mediated High-Density Lipoprotein Maturation Reduces Scavenger Receptor BI Deficiency-Driven Atherosclerosis Susceptibility Despite Unexpected Metabolic Complications.

We tested the hypothesis that enlarged, dysfunctional HDL (high-density lipoprotein) particles contribute to the augmented atherosclerosis susceptibility associated with SR-BI (scavenger receptor BI) deficiency in mice. Approach and Results: We eliminated the ability of HDL particles to fully mature by targeting PLTP (phospholipid transfer protein) functionality. Particle size of the HDL population was almost fully normalized in male and female SR-BI×PLTP double knockout mice. In contrast, the plasma unesterified cholesterol to cholesteryl ester ratio remained elevated. The PLTP deficiency-induced reduction in HDL size in SR-BI knockout mice resulted in a normalized aortic tissue oxidative stress status on Western-type diet. Atherosclerosis susceptibility was-however-only partially reversed in double knockout mice, which can likely be attributed to the fact that they developed a metabolic syndrome-like phenotype characterized by obesity, hypertriglyceridemia, and a reduced glucose tolerance. Mechanistic studies in chow diet-fed mice revealed that the diminished glucose tolerance was probably secondary to the exaggerated postprandial triglyceride response. The absence of PLTP did not affect LPL (lipoprotein lipase)-mediated triglyceride lipolysis but rather modified the ability of VLDL (very low-density lipoprotein)/chylomicron remnants to be cleared from the circulation by the liver through receptors other than SR-BI. As a result, livers of double knockout mice only cleared 26% of the fractional dose of [14C]cholesteryl oleate after intravenous VLDL-like particle injection. We have shown that disruption of PLTP-mediated HDL maturation reduces SR-BI deficiency-driven atherosclerosis susceptibility in mice despite the induction of proatherogenic metabolic complications in the double knockout mice.

Neuropilin-1 Expression on CD4 T Cells Is Atherogenic and Facilitates T Cell Migration to the Aorta in Atherosclerosis.

Neuropilin 1 (Nrp1) is a type I transmembrane protein that plays important roles in axonal guidance, neuronal development, and angiogenesis. Nrp1 also helps migrate thymus-derived regulatory T cells to vascular endothelial growth factor (VEGF)-producing tumors. However, little is known about the role of Nrp1 on CD4 T cells in atherosclerosis. In ApoE-/- mice fed a Western diet for 15 wk, we found a 2-fold increase in Nrp1+Foxp3- CD4 T cells in their spleens, periaortic lymph nodes, and aortas, compared with chow-fed mice. Nrp1+Foxp3- CD4 T cells had higher proliferation potential, expressed higher levels of the memory marker CD44, and produced more IFN-γ when compared with Nrp1- CD4 T cells. Treatment of CD4 T cells with oxLDL increased Nrp1 expression. Furthermore, atherosclerosis-susceptible mice selectively deficient for Nrp1 expression on T cells developed less atherosclerosis than their Nrp1-sufficient counterparts. Mechanistically, we found that CD4 T cells that express Nrp1 have an increased capacity to migrate to the aorta and periaortic lymph nodes compared to Nrp1- T cells, suggesting that the expression of Nrp1 facilitates the recruitment of CD4 T cells into the aorta where they can be pathogenic. Thus, we have identified a novel role of Nrp1 on CD4 T cells in atherosclerosis. These results suggest that manipulation of Nrp1 expression on T cells can affect the outcome of atherosclerosis and lower disease incidence.

Stage-dependent differential effects of interleukin-1 isoforms on experimental atherosclerosis.

Targeting interleukin-1 (IL-1) represents a novel therapeutic approach to atherosclerosis. CANTOS demonstrated the benefits of IL-1β neutralization in patients post-myocardial infarction with residual inflammatory risk. Yet, some mouse data have shown a prominent role of IL-1α rather than IL-1β in atherosclerosis, or even a deleterious effect of IL-1 on outward arterial remodelling in atherosclerosis-susceptible mice. To shed light on these disparate results, this study investigated the effect of neutralizing IL-1α or/and IL-1β isoforms starting either early in atherogenesis or later in ApoE-/- mice with established atheroma. The neutralization of IL-1α or of both IL-1 isoforms impaired outward remodelling during early atherogenesis as assessed by micro-computed tomographic and histologic assessment. In contrast, the neutralization of IL-1β did not impair outward remodelling either during early atherogenesis or in mice with established lesions. Interleukin-1β inhibition promoted a slant of blood monocytes towards a less inflammatory state during atherogenesis, reduced the size of established atheromata, and increased plasma levels of IL-10 without limiting outward remodelling of brachiocephalic arteries. This study established a pivotal role for IL-1α in the remodelling of arteries during early experimental atherogenesis, whereas IL-1β drives inflammation during atherogenesis and the evolution of advanced atheroma in mice.

10,12 Conjugated Linoleic Acid-Driven Weight Loss Is Protective against Atherosclerosis in Mice and Is Associated with Alternative Macrophage Enrichment in Perivascular Adipose Tissue.

The dietary fatty acid 10,12 conjugated linoleic acid (10,12 CLA) promotes weight loss by increasing fat oxidation, but its effects on atherosclerosis are less clear. We recently showed that weight loss induced by 10,12 CLA in an atherosclerosis-susceptible mouse model with characteristics similar to human metabolic syndrome is accompanied by accumulation of alternatively activated macrophages within subcutaneous adipose tissue. The objective of this study was to evaluate whether 10,12 CLA-mediated weight loss was associated with an atheroprotective phenotype. Male low-density lipoprotein receptor deficient (Ldlr−/−) mice were made obese with 12 weeks of a high-fat, high-sucrose diet feeding (HFHS: 36% fat, 36% sucrose, 0.15% added cholesterol), then either continued on the HFHS diet with or without caloric restriction (CR), or switched to a diet with 1% of the lard replaced by either 9,11 CLA or 10,12 CLA for 8 weeks. Atherosclerosis and lipid levels were quantified at sacrifice. Weight loss in mice following 10,12 CLA supplementation or CR as a weight-matched control group had improved cholesterol and triglyceride levels, yet only the 10,12 CLA-treated mice had improved en face and aortic sinus atherosclerosis. 10,12 CLA-supplemented mice had increased lesion macrophage content, with enrichment of surrounding perivascular adipose tissue (PVAT) alternative macrophages, which may contribute to the anti-atherosclerotic effect of 10,12 CLA.

Reduced systemic inflammation and increased reverse cholesterol transport together drive leukocyte ABCA1-mediated protection against atherosclerosis

Aim: Various studies have shown that leukocyte ATP-binding cassette transporter A1 (ABCA1) is an anti-atherogenic factor. It has long been assumed that the increased atherosclerosis susceptibility observed in leukocyte ABCA1 deficient hyperlipidemic mice was due to an accelerated foam cell formation. However, several studies have suggested that leukocyte ABCA1 may also have a beneficial effect on systemic inflammation. In this study we aim to determine the effect of leukocyte ABCA1 deficiency on atherosclerosis susceptibility in mice that are apolipoprotein A1 (apoA1) deficient.

Pannexin1 links lymphatic function to lipid metabolism and atherosclerosis

Extracellular ATP is a central signaling molecule in inflammatory responses. Pannexin1 (Panx1) channels release ATP in a controlled manner and have been implicated in various inflammatory pathologies, but their role in atherogenesis remains elusive. Using atherosclerosis-susceptible mouse models with ubiquitous deletion of Panx1 (Panx1−/−Apoe−/−) or with Cre recombinase-mediated deletion of Panx1 in endothelial cells and monocytes (Tie2-CreTgPanx1fl/flApoe−/−; Panx1delApoe−/−), we identified a novel role for Panx1 in the lymphatic vasculature. Atherosclerotic lesion development in response to high-cholesterol diet was enhanced in Panx1delApoe−/− mice, pointing to an atheroprotective role for Panx1 in endothelial and/or monocytic cells. Unexpectedly, atherogenesis was not changed in mice with ubiquitous Panx1 deletion, but Panx1−/−Apoe−/− mice displayed reduced body weight, serum cholesterol, triglycerides and free fatty acids, suggesting altered lipid metabolism in these Panx1-deficient mice. Mechanistically, Panx1−/−Apoe−/− mice showed impairment of lymphatic vessel function with decreased drainage of interstitial fluids and reduced dietary fat absorption. Thus, the detrimental effect of Panx1 deletion in endothelial and/or monocytic cells during atherogenesis is counterbalanced by an opposite effect resulting from impaired lymphatic function in ubiquitous Panx1-deficient mice. Collectively, our findings unveil a pivotal role of Panx1 in linking lymphatic function to lipid metabolism and atherosclerotic plaque development.

Non-toxic concentrations of cadmium accelerate subendothelial retention of atherogenic lipoproteins in humanized atherosclerosis-susceptible mice

Aim: Cadmium is an important risk factor for cardiovascular disease (CVD). However, the underlying mechanism(s) are unclear. Cadmium has been shown to increase proteoglycan synthesis in vitro. Because proteoglycans mediate a critical step in atherosclerosis by trapping apoB-containing lipoproteins in the artery wall, we hypothesized that cadmium can increase proteoglycan mediated LDL binding and thereby promote subendothelial LDL accumulation. Here we tested the hypothesis that cadmium enhances the subendothelial accumulation of atherogenic lipoproteins in the setting of intimal hyperplasia and modest hypercholesterolemia.

Increased oxidative stress in atherosclerosis-predisposed regions of the mouse aorta

Aims The localization of atherosclerotic lesions to predictable regions in mammalian arteries has been recognized for over a century. We sought to investigate the association between oxidative stress and regional susceptibility of the mouse aorta to atherosclerosis. Main methods En face confocal microscopy was employed to assess oxidative stress in the aortic intima of atherosclerosis-susceptible and protected regions of wild-type C57BL/6 mouse. Expression of reactive oxygen species and antioxidant producing genes were compared in endothelial cells from the susceptible and protected regions. Key findings In vivo administration of redox-sensitive fluorescent dyes revealed an increase in the production of reactive oxygen species (ROS) in the atherosclerosis-susceptible regions relative to the protected regions. In contrast, Hoechst a redox-insensitive dye distributed evenly in the susceptible and protected regions. Accumulation of superoxide in the susceptible regions of the aorta was significantly blocked by the administration of Diphenyleneiodonium, a flavoprotein inhibitor. mRNA levels of superoxide-producing and scavenging enzymes were significantly increased in the regions predisposed to atherosclerosis. The regional difference in oxidative stress was at a lesser magnitude in BALB/c than the atherosclerosis-susceptible mouse (C57BL/6). Significance Our study for the first time demonstrated an augmented oxidative stress in atherosclerosis-susceptible regions of the normal mouse aorta.

Endothelial-Specific Deletion of Connexin40 Promotes Atherosclerosis by Increasing CD73-Dependent Leukocyte Adhesion

Endothelial dysfunction is the initiating event of atherosclerosis. The expression of connexin40 (Cx40), an endothelial gap junction protein, is decreased during atherogenesis. In the present report, we sought to determine whether Cx40 contributes to the development of the disease. Mice with ubiquitous deletion of Cx40 are hypertensive, a risk factor for atherosclerosis. Consequently, we generated atherosclerosis-susceptible mice with endothelial-specific deletion of Cx40 (Cx40del mice). Cx40del mice were indeed not hypertensive. The progression of atherosclerosis was increased in Cx40del mice after 5 and 10 weeks of a high-cholesterol diet, and spontaneous lesions were observed in the aortic sinuses of young mice without such a diet. These lesions showed monocyte infiltration into the intima, increased expression of vascular cell adhesion molecule-1, and decreased expression of the ecto-enzyme CD73 in the endothelium. The proinflammatory phenotype of Cx40del mice was confirmed in another model of induced leukocyte recruitment from the lung microcirculation. Endothelial CD73 is known to induce antiadhesion signaling via the production of adenosine. We found that reducing Cx40 expression in vitro with small interfering RNA or antisense decreased CD73 expression and activity and increased leukocyte adhesion to mouse endothelial cells. These effects were reversed by an adenosine receptor agonist. Cx40-mediated gap junctional communication contributes to a quiescent nonactivated endothelium by propagating adenosine-evoked antiinflammatory signals between endothelial cells. Alteration in this mechanism by targeting Cx40 promotes leukocyte adhesion to the endothelium, thus accelerating atherosclerosis.

Abstract 5064: Endothelial-Specific Deletion of Cx40 Promotes Atherosclerosis by Increasing CD73-Dependent Leukocyte Adhesion

Endothelial dysfunction is the initiating event of atherosclerosis. The expression of connexin40 (Cx40), an endothelial gap junction protein, is decreased during atherogenesis. Here, we sought to determine whether Cx40 contributes to the development of the disease. Mice with ubiquitous deletion of Cx40 are hypertensive, a risk factor for atherosclerosis. Consequently, we generated atherosclerosis-susceptible mice with endothelial-specific deletion of Cx40 (Tie2CRE+ Cx40fl/fl ApoE−/− mice; EC-Cx40del mice). EC-Cx40del mice were indeed not hypertensive. The progression of atherosclerosis was increased in EC-Cx40del mice after 10 weeks high-cholesterol diet; lipid deposition in the thoracic-abdominal aortas was 21.4±3.5% (N=10, p&lt;0.01) in EC-Cx40del mice as compared to 12.6±1.3% for Tie2CRE+ Cx40wt/wt ApoE−/− and 7.1±1.3% for Tie2CRE- Cx40fl/fl ApoE−/− controls. Moreover, spontaneous lesions were observed in the aortic sinuses of young EC-Cx40del mice without diet. These lesions showed monocyte infiltration into the intima, increased expression of VCAM-1 and decreased expression of the ecto-enzyme CD73 in the endothelium. The pro-inflammatory phenotype of EC-Cx40del mice was confirmed in another model of induced leukocyte recruitment from the lung microcirculation. The number of leukocytes that migrated to the alveolar space in response to intratracheal instillation of LPS was markedly enhanced (p&lt;0.001) in EC-Cx40del mice compared to controls, both 3 and 6 hours after the treatment. Endothelial CD73 is known to induce anti-adhesion signaling via the production of adenosine. We found that reducing Cx40 expression in vitro with siRNA or anti-sense decreased CD73 expression and activity, and increased leukocyte adhesion to mouse endothelial cells (p&lt;0.01). These effects were reversed by an adenosine receptor agonist. A control siRNA or Cx40 sense had no effect on leukocyte adhesion. We suggest that Cx40-mediated gap junctional communication contributes to a quiescent non-activated endothelium by propagating adenosine-evoked anti-inflammatory signals between endothelial cells. Alteration in this mechanism by targeting Cx40 promotes leukocyte adhesion to the endothelium, thus accelerating atherosclerosis.

Hyperhomocysteinemia Promotes Inflammatory Monocyte Generation and Accelerates Atherosclerosis in Transgenic Cystathionine β-Synthase–Deficient Mice

Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease. Monocytes display inflammatory and resident subsets and commit to specific functions in atherogenesis. In this study, we examined the hypothesis that HHcy modulates monocyte heterogeneity and leads to atherosclerosis. We established a novel atherosclerosis-susceptible mouse model with both severe HHcy and hypercholesterolemia in which the mouse cystathionine beta-synthase (CBS) and apolipoprotein E (apoE) genes are deficient and an inducible human CBS transgene is introduced to circumvent the neonatal lethality of the CBS deficiency (Tg-hCBS apoE(-/-) Cbs(-/-) mice). Severe HHcy accelerated atherosclerosis and inflammatory monocyte/macrophage accumulation in lesions and increased plasma tumor necrosis factor-alpha and monocyte chemoattractant protein-1 levels in Tg-hCBS apoE(-/-) Cbs(-/-) mice fed a high-fat diet. Furthermore, we characterized monocyte heterogeneity in Tg-hCBS apoE(-/-) Cbs(-/-) mice and another severe HHcy mouse model (Tg-S466L Cbs(-/-)) with a disease-relevant mutation (Tg-S466L) that lacks hyperlipidemia. HHcy increased monocyte population and selective expansion of inflammatory Ly-6C(hi) and Ly-6C(mid) monocyte subsets in blood, spleen, and bone marrow of Tg-S466L Cbs(-/-) and Tg-hCBS apoE(-/-) Cbs(-/-) mice. These changes were exacerbated in Tg-S466L Cbs(-/-) mice with aging. Addition of l-homocysteine (100 to 500 micromol/L), but not l-cysteine, maintained the Ly-6C(hi) subset and induced the Ly-6C(mid) subset in cultured mouse primary splenocytes. Homocysteine-induced differentiation of the Ly-6C(mid) subset was prevented by catalase plus superoxide dismutase and the NAD(P)H oxidase inhibitor apocynin. HHcy promotes differentiation of inflammatory monocyte subsets and their accumulation in atherosclerotic lesions via NAD(P)H oxidase-mediated oxidant stress.

Abstract 3716: Endothelial-specific Deletion Of The Gap Junction Protein Connexin43 Reduces Atherosclerosis In Mice

Decreasing the expression of the gap junction protein connexin43 (Cx43) in mice provides beneficial effects on both the progression and composition of the atherosclerotic lesions. During atherosclerotic lesion development, Cx43 expression is temporarily up-regulated in intimal smooth muscle cells, induced in macrophages close to the lipid core and triggered in a subset of endothelial cells covering the shoulder of atherosclerotic lesions. The purpose of this study was to examine the role of endothelial Cx43 in the development of atherosclerosis in vivo. For this purpose, we have used the Cre-loxP system to create an atherosclerosis-susceptible mouse line in which Cx43 is deleted from the endothelium only. The natural progression of atherosclerosis and the composition of the atherosclerotic lesions were studied in 18 month-old male TIE2Cre+ Cx43fl/fl ApoE−/− mice and TIE2Cre- Cx43fl/fl ApoE−/− littermate controls. Body weights were not different between both groups. Interestingly, the progression of atherosclerosis (measured as % of surface stained with Sudan IV) was significantly reduced in the thoracic-abdominal aorta of TIE2Cre+ Cx43fl/fl ApoE−/− mice (21.53±7.14%; N=4) compared with TIE2Cre- Cx43/fl/fl ApoE−/− controls (46.84±11.60%, N=6, P&lt;0.05). In addition, advanced atheroma in TIE2Cre+ Cx43fl/fl ApoE−/− mice contained less lipids and showed decreased calcium deposition compared with lesions in TIE2Cre- Cx43fl/fl ApoE−/− controls. Thus, endothelial-specific deletion of Cx43 in mice provides beneficial effects on both the natural progression and composition of the atherosclerotic lesions. Future studies are aimed at obtaining insight into the participation of Cx43 in macrophages and smooth muscle cells in atherosclerosis.

The ins and outs of ABCA

Population studies have shown a strong inverse relationship between plasma HDL levels and risk for cardiovascular disease, implying that factors associated with HDL metabolism are cardioprotective. One of these factors is ATP-binding cassette transporter A1 (ABCA1), an integral membrane protein that transports cholesterol and phospholipids to lipid-depleted HDL apolipoproteins, such as apolipoprotein A (apoA)-I (1). Studies of human HDL deficiencies and atherosclerosis-susceptible mouse models have shown that ABCA1 protects against atherogenesis and may prevent disease progression (2–4). These observations have made ABCA1 a promising new therapeutic target for treating cardiovascular disease. Thus, it is critical to understand how ABCA1 functions and is regulated. Despite this strong interest and intensive study over the last 8 years, we still do not have a clear picture of how and where ABCA1 mediates the transfer of cholesterol and phospholipids to apoA-I. Based on structural similarities with other better-characterized ABC transporters, it is assumed by many that ABCA1 pumps lipids to the cell surface where they become accessible for removal by apoA-I. There is evidence, however, that ABCA1bound apoA-I is endocytosed by macrophages and targeted to late endosomal or lysosomal compartments, where it picks up lipids and exports them from cells by an exocytosis process. The concept of this retroendocytosis pathway was introduced by Takahashi and Smith (5) before the discovery of ABCA1. They showed that apoA-I is internalized and resecreted by macrophages in association with cholesterol efflux from cells. Since then, several groups have shown that apoA-I and ABCA1 colocalize in endosomal compartments and that ABCA1 rapidly recycles between intracellular compartments and the plasma membrane (6–8). Cycling of proteins through cells, however, does not imply functional significance. It is well established that cultured cells, particularly macrophages, internalize and resecrete exogenous molecules without any apparent biological effect. In addition, cells rapidly recycle membrane proteins, including those bound to the surface. The possibility that an apoA-I retroendocytosis pathway actually functions to export lipids is supported largely by circumstantial evidence. Cells from subjects with lossof-function mutations in ABCA1 were shown to have defective late endocytotic trafficking and accumulation of cholesterol and phospholipids in late endocytotic vesicles (7), consistent with a role of ABCA1 in removing lipids from these compartments. Trapping ABCA1 in the plasma membrane by cyclosporine treatment was reported to reduce apoA-I-mediated cholesterol efflux (9). Similarly, deletion of a PEST sequence in ABCA1 inhibited its trafficking to late endosomes/lysosomes, and decreased apoA-I-mediated efflux of cholesterol derived from endocytosed and degraded lipoproteins (8). Taken together, these studies support the idea that ABCA1-bound apoA-I traffics to late endosomal vesicles and/or lysosomes, where it forms nascent lipoprotein particles that are subsequently secreted from the cell. There is strong evidence, however, that the ABCA1dependent transfer of cellular lipids to apoA-I occurs at the plasma membrane. Electron microscopy studies have shown that apoA-I selectively interacts with exovesiculated membrane protrusions on the surface of ABCA1expressing cells (10, 11). Inducing ABCA1 in the absence of apolipoproteins increases the amount of cholesterol that appears in plasma membrane domains accessible to the added enzyme cholesterol oxidase, and subsequent addition of apoA-I removes cholesterol exclusively from these domains (12). These findings indicate that ABCA1 can form lipid domains on the cell surface that are accessible for removal by apolipoproteins even before it has contact with apolipoproteins. It has also been shown that partitioning of ABCA1 to the plasma membrane can increase cholesterol efflux under some conditions (8, 13). As reported in the current issue of the Journal of Lipid Research, Faulkner et al. (14) used a three-pronged approach to test the functional relevance of an apoA-I/ ABCA1 retroendocytosis pathway in cultured RAW macrophages. They reasoned that if retroendocytosis plays an important role in apoA-I lipidation, apoA-I should be specifically internalized by cells in response to ABCA1 expression, the mass of apoA-I cycled through the cell should account for most of the lipid efflux, and the degree of apoA-I internalization should correlate with degree of cholesterol efflux. They generated fully functional fluores-

Receptor for Advanced Glycation End Products

The multiligand receptor for advanced glycation end products (RAGE) of the immunoglobulin superfamily is expressed on multiple cell types implicated in the immune-inflammatory response and in atherosclerosis. Multiple studies have elucidated that ligand-RAGE interaction on cells, such as monocytes, macrophages, and endothelial cells, mediates cellular migration and upregulation of proinflammatory and prothrombotic molecules. In addition, recent studies reveal definitive rules for RAGE in effective T lymphocyte priming in vivo. RAGE ligand AGEs may be formed in diverse settings; although AGEs are especially generated in hyperglycemia, their production in settings characterized by oxidative stress and inflammation suggests that these species, in part via RAGE, may contribute to the pathogenesis of atherosclerosis. In murine models of atherosclerosis, vascular inflammation is a key factor and one which is augmented, in parallel with even further increases in RAGE ligands, in diabetic macrovessels. The findings that antagonism and genetic disruption of RAGE in atherosclerosis-susceptible mice strikingly reduces vascular inflammation and atherosclerotic lesion area and complexity link RAGE intimately to these processes and suggest that RAGE is a logical target for therapeutic intervention in aberrant inflammatory mechanisms and in atherosclerosis.

Anatomical differences and atherosclerosis in apolipoprotein E-deficient mice with 129/SvEv and C57BL/6 genetic backgrounds

There are well-known genetic background effects on atherosclerosis susceptibility in mice. To study the basis of these effects, we have generated the apolipoprotein E-null mutation in mouse embryonic stem cells of 129/SvEv origin, maintained it in the inbred strain (129-apoE), and compared these mice with those previously made in strain 129/Ola and backcrossed to a C57BL/6 genetic background (B6-apoE). Plasma cholesterol and triglyceride levels in the apoE-129 mice are twice the levels in apoE-B6, and both VLDL/chylomicron remnants and HDL particles are increased. Regression analysis of plaque size relative to the age of mice suggests that the initiation of atherosclerotic plaque development at the aortic root is slower in 129-apoE mice (intercept at 3.9 months in females and 4.1 months in males) than in B6-apoE mice (1.3 months in females and 2.8 months in males). In contrast, 129-apoE mice develop extensive plaques in the aortic arches earlier than B6-apoE mice. Distinct differences in the geometry of the aortic arch between the two strains suggest that anatomical differences may contribute to the effects of genetic background on atherosclerosis. The 129-apoE/B6-apoE pair thus provides a tool to study factors governing the relation between arterial geometry and the location of plaque development.