Atherosclerosis In ApoE-deficient Mice Research Articles

Adipocyte Fatty Acid-binding Protein Modulates Inflammatory Responses in Macrophages through a Positive Feedback Loop Involving c-Jun NH2-terminal Kinases and Activator Protein-1

Adipocyte fatty acid-binding protein (A-FABP) has emerged as an important mediator of inflammation in macrophages. Macrophage-selective ablation of A-FABP alone is sufficient to prevent the development of high cholesterol diet-induced atherosclerosis in apoE-deficient mice. However, the precise mechanisms whereby A-FABP modulates inflammation remain elusive. Here, we report that A-FABP forms a finely tuned positive loop between JNK and activator protein-1 (AP-1) to exacerbate lipopolysaccharide (LPS)-induced inflammatory responses in macrophages. Real time PCR and luciferase reporter analysis showed that LPS induced A-FABP expression through transcriptional activation. This effect was mediated by JNK, which promoted the recruitment of c-Jun to a highly conserved AP-1 consensus binding motif located within the proximal region of the A-FABP promoter. LPS-induced transactivation of the A-FABP gene was diminished by either pharmacological inhibition of JNK or knocking down c-Jun or by mutating the AP-1 recognition site within the proximal region (-122 to -116 bp) of the A-FABP promoter. Conversely, the LPS-evoked phosphorylation of JNK, activation of AP-1, and production of pro-inflammatory cytokines were markedly attenuated by pharmacological or genetic suppression of A-FABP in macrophages. Furthermore, the LPS-induced elevation in A-FABP expression could also be prevented by the selective A-FABP inhibitor BMS309403. These findings support the notion that pharmacological inhibition of A-FABP represents a valid strategy for treating inflammation-related disorders such as atherosclerosis.

Dietary coenzyme Q10 does not protect against cigarette smoke-augmented atherosclerosis in apoE-deficient mice

Dietary coenzyme Q10 reduces spontaneous atherosclerosis in the apoE-deficient mouse model of experimental atherosclerosis. We have shown previously that exposure to sidestream cigarette smoke (SSCS) enhances atherosclerotic lesion formation in apoE-deficient mice. The aim of the present study was to determine if CoQ10 protected against SSCS-mediated atherosclerosis. Female apoE-deficient mice were fed a saturated fat-enriched diet (SFD) alone, or supplemented with 1% wt/wt coenzyme Q10 (SFD-Q10). Mice in each diet group were exposed to SSCS for 4 hrs/day, 5 days/week in a whole-body exposure chamber maintained at 35 ± 4 mg smoke particulates/m 3. Mice kept in filtered ambient air served as controls. Mice were euthanized after either 6 or 15 weeks of SSCS exposure and following measurements were performed: i) lung 7-ethoxyresorufin-O-deethylase (EROD) activity; ii) plasma cholesterol and CoQ10 concentrations; iii) aortic intimal area covered by atherosclerotic lesions; and, iv) pathological characterization of lesions. Lung EROD activity increased in SSCS mice of both diet groups, confirming SSCS exposure. Plasma concentrations of CoQ10 in SFD-Q10-fed mice were increased markedly in comparison to SFD-fed mice. Plasma cholesterol concentrations and distributions of cholesterol in lipoprotein fractions were unaffected by SSCS exposure. Dietary supplementation with CoQ10 significantly reduced atherosclerotic lesions in control mice. As reported previously, exposure to SSCS increased the size of lesions in apoE-/- mice at both time points. However, dietary supplementation with CoQ10 had no effect on atherosclerotic lesions augmented by SSCS exposure. The results suggest a role of oxidative processes in smoke-augmented atherosclerosis that are different than those mitigated by CoQ10.

Cannabinoid 1 Receptor Blockade Reduces Atherosclerosis with Enhances Reverse Cholesterol Transport

A recent clinical study using coronary intravascular ultrasound showed that rimonabant, a cannabinoid 1 (CB1) receptor antagonist, significantly reduced total atheroma volume, suggesting that CB1 receptor blockade could be beneficial in anti-atherogenic therapy. The reverse cholesterol transport (RCT) system plays important roles in atherogenesis. We investigated whether CB1 receptor blockade could modulate atherogenesis in mice. Oral administration of rimonabant (8 mg/kg/day) to apolipoprotein E-deficient mice for 3 months significantly reduced the relative area of atherosclerotic lesions in the aorta (vehicle; 12.6+/-4.0% vs. rimonabant; 9.7+/-2.3, n=12 each, p<0.05) with an increase in serum adiponectin levels (15.6+/-2.3 microg/mL vs. 12.2+/-2.1, n=12 each, p<0.001), without affecting body weight or serum cholesterol levels. Rimonabant tended to increase serum high-density lipoprotein cholesterol (HDL-C) (p=0.05). The relative area of atherosclerotic lesions in the aorta correlated inversely with serum HDL-C levels (r=-0.45, n=24, p<0.05). Rimonabant upregulated the mRNA expression levels of various components of the RCT system on THP-1 cell-derived macrophages (scavenger receptor B1: 1.15+/-0.12 fold, n=6; p<0.05, ATP-binding cassette [ABC] transporter G1: 1.23+/-0.11 fold, n=6; p<0.01), but not ABCA1 (1.13+/-0.20 fold, n=6; p=0.13). CB1 receptor blockade reduced atherosclerosis in apoE-deficient mice through an increase in serum adiponectin levels and activation of the RCT system. CB1 receptor blockade may be therapeutically beneficial for atherogenesis by increasing the serum adiponectin level and enhancing of the RCT system.

Differential effects of medroxyprogesterone acetate on thrombosis and atherosclerosis in mice

The risk for cardiovascular events including venous and arterial disease and stroke is elevated after treatment with estrogen and medroxyprogesterone acetate (MPA) in postmenopausal women. Here, we have investigated the effect of MPA on arterial thrombosis and atherosclerosis in a murine model of atherosclerosis. Apolipoprotein E (ApoE)-/- mice were bilaterally ovariectomized and treated with placebo, MPA (27.7 microg day(-1)) and MPA + 17-beta-oestradiol (E2; 1.1 microg day(-1)) for 90 days, on a Western-type diet. Thrombotic response was measured in a photothrombosis model, platelet activation by fluorescence activated cell sorting (FACS) analysis (CD62P) and thrombin generation by the endogenous thrombin potential (ETP). Furthermore, aortic plaque burden and aortic root plaque composition were determined. MPA and MPA + E2-treated animals showed an aggravated thrombotic response shown by significantly reduced time to stable occlusion. The pro-thrombotic effect of MPA was paralleled by increased ETP whereas platelet activation was not affected. Furthermore, MPA + E2 reduced the number of cells positive for alpha-smooth muscle actin and increased hyaluronan in the plaque matrix. Interestingly, total plaque burden was reduced by MPA but unchanged by MPA + E2. Long-term treatment with MPA and MPA + E2 increased arterial thrombosis despite inhibitory effects of MPA on atherosclerosis in ApoE-deficient mice. Increased thrombin formation, reduced smooth muscle content and remodelling of non-collagenous plaque matrix may be involved in the pro-thrombotic effects. Thus, MPA exhibits differential effects on arterial thrombosis and on atherosclerosis.

Abstract 5070: Pitavastatin Has Anti-atherosclerotic Effects via Inhibition of TRAIL Expression on CD4 T Cells

Background: Statins reduce cardiovascular-related morbidity and mortality, however their effects on inflammation in atherosclerosis are not fully understood. We investigated whether pitavastatin can modulate cytotoxic functions of CD4 T cells in acute coronary syndrome (ACS) and inhibit the development of atherosclerosis in apoE deficient mice (apoE KO). Methods and Results: Fresh CD4 T cells were isolated from blood obtained from 37 patients with ACS without statin treatment on admission and 34 control patients. CD4 T cells derived from ACS patients effectively induced vascular endothelial cell (EC) apoptosis significantly more than controls ( P &lt;0.0001). CD4 T cells from ACS patients expressed higher levels of TRAIL ( P &lt;0.002) than those of controls. We further examined whether statin could regulate TRAIL expression and cytotoxicity of CD4 T cells. Pitavastatin (PIT, 10 μ M) blocked CD4 T cell-mediated EC apoptosis ( P =0.001) and reduced the expression of TRAIL ( P &lt;0.01) on CD4 T cells from ACS patients. Finally, to investigate whether TRAIL-expressing CD4 T cells had biological relevance in vivo, 12-week-old ApoE-deficient mice were fed a high-fat diet supplemented with 1mg/kg/day PIT (n= 5), 10mg/kg/day (n= 5), or Vehicle (0.5% carboxy-methyl cellulose, n= 5) for 8 weeks. Sudan IV staining of the aorta and oil red O-staining of the aortic sinus revealed that 1mg/kg/day and 10mg/kg/day PIT significantly suppressed the development of atherosclerotic lesions depending on dose ( P &lt;0.05, P &lt;0.0001, respectively). In addition, PIT suppressed inflammatory infiltrates and CD4 T cells in atherosclerotic plaque by immunohistochemistry. Furthermore, to investigate whether PIT inhibited the expression of TRAIL on CD4 T cells, CD4 T cells were isolated from spleen and analyzed by FACS. The expression of TRAIL was significantly inhibited by 1mg/kg/day and 10mg/kg/day PIT ( P &lt;0.01, P &lt;0.005, respectively). Conclusions: TRAIL-expressing CD4 T cells are enriched in ACS patients and apoE deficient mice and induce endothelial injury, which may contribute to the increasing plaque instability. Pitavastatin therapy may prevent T cell-mediated endothelial cell damage and contribute to the prevention of atherosclerotic plaque development and plaque instability.

PPARγ1-Induced Caveolin-1 Enhances Cholesterol Efflux and Attenuates Atherosclerosis in Apolipoprotein E-Deficient Mice

Objective: Caveolin-1 (Cav-1) may positively or negatively influence the development of atherosclerosis, depending on the cell type and the metabolic pathways regulated by this protein. We investigate the effects of Cav-1 on cholesterol efflux in RAW264.7 infected with AdPPARγ1 and whether Cav-1 could attenuate established atherosclerotic lesions in PPARγ1-treated apoE-deficient mice. Methods and Results: Compared with AdGFP control, PPARγ1 and Cav-1 were constitutively overexpressed in AdPPARγ1-infected RAW264.7 cells, which stimulated cholesterol efflux to apolipoprotein A-I. Using a small interfering RNA approach (Cav-1-siRNA) we achieved an efficient and specific knockdown of caveolin-1 expression (80%), which resulted in a remarkable reduction of cholesterol efflux in RAW264.7 cells . Moreover, PPARγ1-treated Cav-1-siRNA RAW264.7 cells showed more ability to stimulate cholesterol efflux than Cav-1-siRNA RAW264.7 cells, but far less than control-siRNA RAW264.7 cells and PPARγ1-treated RAW264.7 cells. In addition, 40-week-old apoE-deficient mice fed a Western-type diet and infected for 4 weeks with AdPPARγ1 showed induced Cav-1 expression in aortic vascular endothelial cells, smooth muscle cells and macrophages, as well as attenuated established atherosclerotic lesions. Conclusions: PPARγ1 gene therapy could induce Cav-1 expression and enhance cholesterol efflux and attenuate atherosclerosis in apoE-deficient mice.

Structural and functional consequences of the Milano mutation (R173C) in human apolipoprotein A-I

Carriers of the apolipoprotein A-I(Milano) (apoA-I(M)) variant, R173C, have reduced levels of plasma HDL but no increase in cardiovascular disease. Despite intensive study, it is not clear whether the removal of the arginine or the introduction of the cysteine is responsible for this altered functionality. We investigated this question using two engineered variations of the apoA-I(M) mutation: R173S apoA-I, similar to apoA-I(M) but incapable of forming a disulfide bond, and R173K apoA-I, a conservative mutation. Characterization of the lipid-free proteins showed that the order of stability was wild type approximately R173K>R173S>R173C. Compared with wild-type apoA-I, apoA-I(M) had a lower affinity for lipids, while R173S apoA-I displayed intermediate affinity. The in vivo effects of the apoA-I variants were measured by injecting apoA-I-expressing adeno-associated virus into apoA-I-null mice. Mice that expressed the R173S variant again showed an intermediate phenotype. Thus, both the loss of the arginine and its replacement by a cysteine contribute to the altered properties of apoA-I(M). The arginine is potentially involved in an intrahelical salt bridge with E169 that is disrupted by the loss of the positively charged arginine and repelled by the cysteine, destabilizing the helix bundle domain in the apoA-I molecule and modifying its lipid binding characteristics.

The emerging roles of PAF acetylhydrolase

Platelet-activating factor (PAF), a phospholipid autacoid with potent effects throughout the innate immune system, is selectively degraded by two small families of PAF acetylhydrolases (PAF-AHs). These Ca2+-independent phospholipases A2 display remarkable specificity for the length of the sn-2 residue, but this selectivity is lost as the residue gains oxygen functions. Two of the PAF-AHs therefore are specific oxidized phospholipid phospholipases that reduce inflammation, but also remove oxidatively truncated phospholipids that induce apoptosis. The roles of these enzymes are manifold, and their separate and combined functions are now being addressed in model systems and clinical studies.

Apolipoprotein E knock-out and knock-in mice: atherosclerosis, metabolic syndrome, and beyond

Given the multiple differences between mice and men, it was once thought that mice could not be used to model atherosclerosis, principally a human disease. Apolipoprotein E-deficient (apoEKO) mice have convincingly changed this view, and the ability to model human-like plaques in these mice has provided scientists a platform to study multiple facets of atherogenesis and to explore potential therapeutic interventions. In addition to its well-established role in lipoprotein metabolism, recent observations of reduced adiposity and improved glucose homeostasis in apoEKO mice suggest that apoE may also play a key role in energy metabolism in peripheral organs, including adipose tissue. Finally, along with apoEKO mice, knockin mice expressing human apoE isoforms in place of endogenous mouse apoE have provided insights into how quantitative and qualitative genetic alterations interact with the environment in the pathogenesis of complex human diseases.

Mechanisms and consequences of macrophage apoptosis in atherosclerosis

Macrophage apoptosis is an important feature of atherosclerotic plaque development. Research directed at understanding the functional consequences of macrophage death in atherosclerosis has revealed opposing roles for apoptosis in atherosclerotic plaque progression. In early lesions, macrophage apoptosis limits lesion cellularity and suppresses plaque progression. In advanced lesions, macrophages apoptosis promotes the development of the necrotic core, a key factor in rendering plaques vulnerable to disruption and in acute lumenal thrombosis. The first section of this review will examine the role of phagocytic clearance of apoptotic macrophages, a process known as efferocytosis, in the dichotomous roles of macrophage apoptosis in early vs. advanced lesions. The second section will focus on the molecular and cellular mechanisms that are thought to govern macrophage death during atherosclerosis. Of particular interest is the complex and coordinated role that the endoplasmic reticulum (ER) stress pathway and pattern recognition receptors (PRRs) may play in triggering macrophage apoptosis.

VLDL best predicts aortic root atherosclerosis in LDL receptor deficient mice

Hyperlipidemia is a major risk factor for developing atherosclerosis in humans, and epidemiological studies have correlated specific lipoprotein levels with cardiovascular disease risk. Murine models of atherosclerosis rely on the induction of hyperlipidemia for vascular lesions to form, but the pathogenic contributions attributed to different lipoprotein populations are not well defined. To address this issue, we analyzed over 300 LDL receptor (LDLR) deficient mice that have been fed a high-fat diet and for which a full lipoprotein profile and aortic root atherosclerosis values were assessed. Overall, aortic root atherosclerosis is best predicted by plasma VLDL cholesterol levels with less predictive value derived from either LDL or HDL cholesterol. Triglyceride levels are more atherogenic in female mice, especially immune competent females, and depletion of the adaptive immune system leads to a global reduction in plasma lipid levels and aortic root lesion size yet does not appear to alter the atherogenic potential of individual lipoprotein subspecies. In contrast, HDL-cholesterol is a better predictor of aortic root atherosclerosis in apoE-deficient mice. In summary, this large scale analysis of high-fat diet fed LDLR deficient mice highlight the relationship between different plasma lipid components, especially VLDL-cholesterol, and aortic root atherosclerosis.

Anti-Atherosclerotic Effects of Dihomo-.GAMMA.-Linolenic Acid in ApoE-Deficient Mice

Dihomo-gamma-linolenic acid (DGLA) is an n-6 polyunsaturated fatty acid that is mainly metabolized to an anti-inflammatory eicosanoid, prostaglandin (PG) E1, via the cyclooxygenase (COX) pathway. We evaluated the effect of DGLA on atherosclerosis in apoE-deficient mice and studied the mechanism of the anti-atherosclerotic effect. ApoE-deficient mice were fed a normal diet supplemented with 0.5% DGLA or vehicle for 6 months. ApoE-deficient mice were also fed a high-cholesterol diet supplemented with 0.5% DGLA or vehicle for 1 month. To clarify the influence of a COX inhibitor, naproxen, on the anti-atherosclerotic effect of DGLA, age-matched apoE-deficient mice fed a high-cholesterol diet supplemented with 0.5% DGLA were given oral naproxen for 1 month. In normal diet-fed mice, acetylcholine-induced vascular relaxation was significantly greater in the DGLA group than in the vehicle group. NADPH oxidase subunits, p22phox and gp91phox, intercellular adhesion molecule-1, and vascular cellular adhesion molecule-1 were significantly lower in the DGLA group than in the vehicle group, and DGLA significantly prevented atherosclerosis. In high-cholesterol diet-fed mice, DGLA also significantly prevented atherosclerosis, but the anti-atherosclerotic effect was attenuated by naproxen. DGLA may have an anti-atherosclerotic effect in apoE-deficient mice via PGE1 formation.

TLR/MyD88 and Liver X Receptor α Signaling Pathways Reciprocally Control Chlamydia pneumoniae-Induced Acceleration of Atherosclerosis

Experimental and clinical studies link Chlamydia pneumoniae infection to atherogenesis and atherothrombotic events, but the underlying mechanisms are unclear. We tested the hypothesis that C. pneumoniae-induced acceleration of atherosclerosis in apolipoprotein E (ApoE)(-/-) mice is reciprocally modulated by activation of TLR-mediated innate immune and liver X receptor alpha (LXRalpha) signaling pathways. We infected ApoE(-/-) mice and ApoE(-/-) mice that also lacked TLR2, TLR4, MyD88, or LXRalpha intranasally with C. pneumoniae followed by feeding of a high fat diet for 4 mo. Mock-infected littermates served as controls. Atherosclerosis was assessed in aortic sinuses and in en face preparation of whole aorta. The numbers of activated dendritic cells (DCs) within plaques and the serum levels of cholesterol and proinflammatory cytokines were also measured. C. pneumoniae infection markedly accelerated atherosclerosis in ApoE-deficient mice that was associated with increased numbers of activated DCs in aortic sinus plaques and higher circulating levels of MCP-1, IL-12p40, IL-6, and TNF-alpha. In contrast, C. pneumoniae infection had only a minimal effect on atherosclerosis, accumulation of activated DCs in the sinus plaques, or circulating cytokine increases in ApoE(-/-) mice that were also deficient in TLR2, TLR4, or MyD88. However, C. pneumoniae-induced acceleration of atherosclerosis in ApoE(-/-) mice was further enhanced in ApoE(-/-)LXRalpha(-/-) double knockout mice and was accompanied by higher serum levels of IL-6 and TNF-alpha. We conclude that C. pneumoniae infection accelerates atherosclerosis in hypercholesterolemic mice predominantly through a TLR/MyD88-dependent mechanism and that LXRalpha appears to reciprocally modulate and reduce the proatherogenic effects of C. pneumoniae infection.

Diversity of Denizens of the Atherosclerotic Plaque

The atherosclerotic plaque typically harbors cells of several lineages whose conversations, mediated by extracellular or cell surface–associated messengers, influence decisively the biology and clinical consequences of the lesion. Early vascular biology studies defined the resting state of the endothelium, characterized by the elaboration of antithrombotic and vasodilatory mediators. The activated endothelium recruits inflammatory leukocytes, favors clot accumulation, participates in angiogenesis, and can influence the behavior of subjacent smooth muscle cells in ways that favor atherogenesis and vasoconstriction (Figure). More recently, we have come to appreciate that the endothelial cell not only can exhibit a spectrum of functions, but that some may arise postnatally from bone marrow–derived precursors.1 Thus, the heterogeneity of endothelial cells depends not only on the mutability of their function but also on their origin. The diversity of endothelium depends not only on lineage but also location, with increasingly well-understood differences between arterial, microvascular, and venous endothelial cells. Figure. Heterogeneity of major cell types in atherosclerotic plaques. Vascular biologists have long recognized heterogeneity of endothelial cells and smooth muscle cells, now understood to result from local mediator milieu, biomechanical stimuli, and different embryological origins. Indeed, recent data suggest that both of these intrinsic vascular cell types can arise in postnatal life from bone marrow–derived precursors. Immunological dogma recognizes several T-cell populations, exemplified here by the Th1 and Th2 subsets, which on the balance exert opposing influences on atherogenesis. New data now establish the relevance to atherosclerosis and hyperlipidemia of monocyte heterogeneity. Monocytes that bear high levels of the markers Ly6c/Gr-1 and P-selectin glycoprotein ligand exhibit more proinflammatory functions than their …

Inflammation and Cardiovascular Disease

Inflammation plays an important role in the development and progression of a variety of cardiovascular conditions, most notably coronary atherosclerosis and congestive heart failure.1,2 A number of inflammatory molecules have been implicated in these processes, including interleukin-1 (IL-1). The IL-1 gene family consists of 3 proteins, IL-1α, IL-1β, and IL-1 receptor antagonist (IL-1ra). IL-1α and IL-1β exert their similar effects by binding to the IL-1 type I receptor. The IL-1 type II receptor also binds IL-1α and IL-1β but acts as a decoy receptor and is not involved in signal transduction, thereby counterbalancing the inflammatory effects of IL-1α and IL-1β. IL-1ra is an endogenous inhibitor of IL-1α and IL-1β, which competitively binds to the IL-1 type I receptor without activating it.3 Article p 2662 Both IL-1 and IL-1ra are produced by endothelial cells, smooth muscle cells, and macrophages. IL-1 secretion is induced by microbial products that stimulate toll-like receptors and by certain endogenous triggers, such as uric acid produced during cell death. Both types of agonists stimulate a cytosolic complex of proteins termed the inflammasome, which activates caspase-1 to enable secretion of IL-1β. The potential of this IL-1β pathway for systemic inflammation is demonstrated not only by gout but also by the clinical effects of activating mutations in cryopyrin (also known as NALP3 or CIAS1), one of the inflammasome components, which causes familial cold autoinflammatory syndrome, Muckle-Wells syndrome, and neonatal-onset multisystem inflammatory disease.4 Circulating levels of IL-1 are associated with the presence of traditional cardiac risk factors, such as diabetes mellitus, hypertension, smoking, and dyslipidemia. Elevated levels of IL-1 result in secretion of chemokines and other cytokines (eg, IL-6), increased expression of adhesion molecules, activation of endothelial and smooth muscle cell proliferation, macrophage activation, and increased vascular permeability. This cascade promotes atherosclerosis and plaque destabilization. IL-1 and …

CD44 Expressed on Both Bone Marrow–Derived and Non–Bone Marrow–Derived Cells Promotes Atherogenesis in ApoE-Deficient Mice

The purpose of this study was to distinguish the contributions of CD44 expressed on bone marrow-derived and non-bone marrow-derived cells to atherosclerosis. Using bone marrow chimeras, we compared the contributions of CD44 expressed on bone marrow-derived cells versus non-bone marrow-derived cells to the vascular inflammation underlying atherosclerosis. We show that CD44 in both bone marrow-derived and non-bone marrow-derived compartments promotes atherosclerosis in apoE-/- mice and mediates macrophage and T cell recruitment to lesions in vivo. We also demonstrate that CD44 on endothelial cells (ECs) as well as on macrophages and T cells enhances leukocyte-endothelial cell adhesion and transendothelial migration in vitro. Furthermore, CD44 on vascular smooth muscle cells (VSMCs) regulates their hyaluronan (HA)-dependent migration. Interestingly, in mice lacking CD44 in both compartments, where we observed the least inflammation, we also observed enhanced fibrous cap formation. CD44 expressed on bone marrow-derived and non-bone marrow-derived cells both promote atherosclerosis in apoE-deficient mice. Furthermore, CD44 plays a pivotal role in determining the balance between inflammation and fibrosis in atherosclerotic lesions which can impact clinical outcome in humans.

Role of cathepsin K in structural changes in brachiocephalic artery during progression of atherosclerosis in apoE-deficient mice

Cathepsin K, a potent extracellular matrix degrading cysteine protease, has been linked to the pathogenesis of osteoporosis, arthritis, cardiovascular and respiratory diseases. Here, we report the effects of cathepsin K deficiency (ctsK−/−) on atherosclerotic plaque formation in brachiocephalic arteries in an aggressive atherosclerosis model using apoE-deficient mice on cholate-containing high fat diet (HFD). On this diet, apoE−/− mice displayed severe lesions with buried fibrous caps after 8 weeks, whereas the apoE−/−ctsK−/− mice revealed a significantly decreased number of buried fibrous caps accompanied by increased collagen content in plaque areas and fibrous cap thickness. After 16 weeks of HFD, ctsK−/− mice had smaller plaque areas and maintained the structure of the tunica media in terms of their smooth muscle cell content and elastic lamina integrity. Overall macrophage content in the tunica media was lower in ctsK−/− mice but higher in the plaque area after 8 weeks of HFD. Decreased apoptosis rates in atherosclerotic plaques in brachiocephalic arteries of cathepsin K-deficient indicated a lower level of inflammation. In conclusion, cathepsin K deficiency appears to increase lesion stability in brachiocephalic arteries by maintaining the integrity of the tunica media and by decreasing plaque vulnerability to rupture.

15-Lipoxygenase-1 in the Vasculature

See related article, pages 177–184 Lipoxygenases (LOs) constitute a heterogeneous family of enzymes that catalyze the stereoselective dioxygenation of polyunsaturated fatty acids to their corresponding hydroperoxy derivatives.1,2 In mammals, LOs are categorized with respect to their positional specificity of arachidonic acid oxygenation into 5-, 8-, 12-, and 15-LOs.1–3 These enzymes induce structural and metabolic changes in cells during a wide variety of physiological and pathological processes, such as differentiation, carcinogenesis, inflammation, and atherogenesis. Accumulating studies have suggested diverse and opposing roles for the various LO pathways in the pathogenesis of human diseases, particularly cancer and atherosclerosis.2–5 Consistent with the existence of multiple isoforms of LOs, a variety of intermediate and end products of arachidonic acids are found in various cell types, and they in turn activate diverse signaling cascades, resulting in diverse outcomes. Therefore, sophisticated understanding of the expression pattern of individual isozymes in the target cells and biological actions of the corresponding metabolites should be prerequisite for predicting their roles in disease processes. Among the 4 mammalian LO subfamilies, 15-lipoxygenase-1 (also known as 12/15-lipoxygenase in mice) catalyzes the transformation of free arachidonic acid to 12-hydroperoxy-eicosatetraenoic acid and15-hydroperoxyeicosatetraenoic acid and the transformation of linoleic acid to 13-hydroperoxyoctadecadienoic acid (13-HPODE) (the reduced product is 13-HODE).3,4 Both human 15-LO-1 and mouse 12/15-LO enzymes also metabolize more complex lipids, including phospholipids, cholesterol esters, and plasma lipoproteins. 15-LO-1 was considered initially to be the reticulocyte LO that can react with mitochondrial membrane lipids,6 but subsequently, a constitutive expression of 15-LO-1 has been reported in various types of cells, such as immature red blood cells, eosinophils, and airway epithelial cells.3 In addition, the eicosanoid products of 12/15-LO have been detected in vascular cells, and 15-LO-1 protein has been shown to be localized to aortic atherosclerotic lesions in rabbits …

Additive anti-atherogenic effect of thromboxane receptor antagonism with 12/15lipoxygenase gene disruption in apolipoprotein E-deficient mice

Previous studies in mouse models showed that 12/15lipoxygenase (12/15LO) gene disruption diminishes atherosclerosis. Pharmacologic suppression of thromboxane (Tx) A 2 biosynthesis or blockade of its receptor also reduces the development of the disease in the same models. We tested the hypothesis that simultaneous genetic absence of 12/15LO with TxA 2 receptor blockade might result in an additive anti-atherogenic effect. Apolipoprotein E (apoE)-deficient mice and apoE-deficient mice lacking 12/15LO were maintained on normal chow diet, or chow supplemented with BM-573, a selective TxA 2 receptor antagonist, for 12 weeks. Urinary TxA 2 and prostacyclin metabolites, isoprostaneF 2α-III and atherosclerotic aortic lesions were assessed. 12/15LO gene disruption resulted in significantly reduced atherosclerotic lesion areas and decreased urinary isoprostaneF 2α-III in apoE-deficient mice. TxA 2 receptor antagonism alone also afforded a significant reduction in atherosclerosis in apoE-deficient mice. However, thromboxane receptor blockade resulted in an additive and more potent anti-inflammatory and anti-atherogenic effect when administered to apoE-deficient mice lacking 12/15LO. These results suggest that the 12/15LO- and TxA 2 receptor-mediated pro-atherogenic effects are two distinct pathways and represent two separate therapeutic targets for a better anti-atherogenic strategy.

Effect of chronic treatment with acetylsalicylic acid and clopidogrel on atheroprogression and atherothrombosis in ApoE-deficient mice in vivo

Acetylsalicylic acid (ASA) and the thienopyridine clopidogrel are established anti-platelet drugs that significantly reduce secondary cardiovascular events in patients with manifest atherosclerosis. However, their impact on atherosclerotic lesion development remains controversial. Four-week-old ApoE-deficient mice were randomly assigned to four groups receiving a cholesterol diet together with either ASA (5 mg/kg), or clopidogrel (25 mg/kg), or a combination of both ASA and clopidogrel, or vehicle for 8-12 weeks. Using intravital microscopy we found that daily administration of ASA in combination with clopidogrel reduces platelet thrombus formation following rupture of atherosclerotic plaque in vivo by approximately 50%. However, therapy with ASA or clopidogrel alone, or in combination for a period of 8-12 weeks had no significant effect on adhesion of platelets to dysfunctional endothelial cells or on atherosclerotic lesion formation in the aortic root or the carotid artery. In conclusion, anti-platelet therapy is effective in reducing platelet adhesion and subsequent thrombus formation following rupture of atherosclerotic plaque in vivo. However, our data do not support a role of either drug in the primary prevention of atherosclerosis in ApoE-deficient mice.