Role of renin-angiotensin system in activation of macrophages by modified lipoproteins.

Angiotensin II favors the development of atherosclerosis. Our goal was to determine if foam cell formation increases angiotensin II generation by the endogenous renin-angiotensin system (RAS) and if endogenously produced angiotensin II promotes lipid accumulation in macrophages. Differentiated THP-1 cells were treated with acetylated low-density lipoproteins (ac-LDL), native LDL (n-LDL), or no LDL. Expression of RAS genes was assessed and angiotensin I/II levels were quantified in media and cell lysate. Ac-LDL increased angiotensin I/II levels and the angiotensin II/I ratio in cells and media after foam cell formation. Renin mRNA or activity did not change, but renin blockade completely inhibited the increase in angiotensin II. Angiotensinogen mRNA but not protein level was increased. Angiotensin-converting enzyme (ACE) and cathepsin G mRNA and activities were enhanced by ac-LDL. Inhibition of renin, ACE, or the angiotensin II receptor 1 (AT1-receptor) largely abolished cholesteryl ester formation in cells exposed to ac-LDL and decreased scavenger receptor A (SR-A) and acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT-1) protein levels. Inhibition of renin or the AT1-receptor in cells treated with oxidized LDL also decreased SR-A and ACAT-1 protein and foam cell formation. ac-LDL also increased angiotensin II by human peripheral blood monocyte-derived macrophages, whereas blockade of renin decreased cholesterol ester formation in these macrophages. These findings indicate that, during foam cell formation, angiotensin II generation by the endogenous RAS is stimulated and that endogenously generated angiotensin II is crucial for cholesterol ester accumulation in macrophages exposed to modified LDL.

Cathepsin G deficiency decreases complexity of atherosclerotic lesions in apolipoprotein E-deficient mice.

Cathepsin G is a serine protease with a broad range of catalytic activities, including production of angiotensin II, degradation of extracellular matrix and cell-cell junctions, modulation of chemotactic responses, and induction of apoptosis. Cathepsin G mRNA expression is increased in human coronary atheroma vs. the normal vessel. To assess whether cathepsin G modulates atherosclerosis, cathepsin G knockout (Cstg(-/-)) mice were bred with apolipoprotein E knockout (Apoe(-/-)) mice to obtain Ctsg(+/-)Apoe(-/-) and Ctsg(+/+)Apoe(-/-) mice. Heterozygous cathepsin G deficiency led to a 70% decrease in cathepsin G activity in bone marrow cells, but this reduced activity did not impair generation of angiotensin II in bone marrow-derived macrophages (BMDM). Atherosclerotic lesions were compared in male Cstg(+/-)Apoe(-/-) and Cstg(+/+)Apoe(-/-) mice after 8 wk on a high-fat diet. Plasma cholesterol levels and cholesterol distribution within serum lipoprotein fractions did not differ between genotypes nor did the atherosclerotic lesion areas in either the aortic root or aortic arch. Cstg(+/-)Apoe(-/-) mice, however, showed a lower percentage of complex lesions within the aortic root and a smaller number of apoptotic cells compared with Cstg(+/+)Apoe(-/-) littermates. Furthermore, apoptotic Cstg(-/-) BMDM were more efficiently engulfed by phagocytic BMDM than were apoptotic Ctsg(+/+) BMDM. Thus cathepsin G activity may impair efferocytosis, which could lead to an accumulation of lesion-associated apoptotic cells and the accelerated progression of early atherosclerotic lesions to more complex lesions in Apoe(-/-) mice.

Naringenin decreases progression of atherosclerosis by improving dyslipidemia in high-fat-fed low-density lipoprotein receptor-null mice.

OBJECTIVE: Naringenin is a citrus flavonoid that potently inhibits the assembly and secretion of apolipoprotein B100-containing lipoproteins in cultured hepatocytes and improves the dyslipidemia and insulin resistance in a mouse model of the metabolic syndrome. In the present study, we used low-density lipoprotein receptor-null mice fed a high-fat diet (Western, TD96125) to test the hypothesis that naringenin prevents atherosclerosis. METHODS AND RESULTS: Three groups (chow, Western, and Western plus naringenin) were fed ad libitum for 6 months. The Western diet increased fasting plasma triglyceride (TG) (5-fold) and cholesterol (8-fold) levels compared with chow, whereas the addition of naringenin significantly decreased both lipids by 50%. The Western-fed mice developed extensive atherosclerosis in the aortic sinus because plaque area was increased by 10-fold compared with chow-fed animals. Quantitation of fat-soluble dye (Sudan IV)-stained aortas, prepared en face, revealed that Western-fed mice also had a 10-fold increase in plaque deposits throughout the arch and in the abdominal sections of the aorta, compared with chow. Atherosclerosis in both areas was significantly decreased by more than 70% in naringenin-treated mice. Consistent with quantitation of aortic lesions, the Western-fed mice had a significant 6-fold increase in cholesterol and a 4-fold increase in TG deposition in the aorta compared with chow-fed mice. Both were reduced more than 50% by naringenin. The Western diet induced extensive hepatic steatosis, with a 10-fold increase in both TG and cholesteryl ester mass compared with chow. The addition of naringenin decreased both liver TG and cholesteryl ester mass by 80%. The hyperinsulinemia and obesity that developed in Western-fed mice was normalized by naringenin to levels observed in chow-fed mice. CONCLUSIONS: These in vivo studies demonstrate that the citrus flavonoid naringenin ameliorates the dyslipidemia in Western-fed low-density lipoprotein receptor-null mice, leading to decreased atherosclerosis; and suggests a potential therapeutic strategy for the hyperlipidemia and increased risk of atherosclerosis associated with insulin resistance.

Specific loss of toll-like receptor 2 on bone marrow derived cells decreases atherosclerosis in LDL receptor null mice.

Innate immunity and, notably, Toll-like receptors (TLR), have an important role in atherogenesis. We have tested the hypothesis that the selective loss of TLR-2 by cells of bone marrow (BM) origin will protect low-density receptor-deficient (Ldlr (-/-)) mice from both early- and late-stage atherosclerosis. BM cells from Tlr2(+/+) and Tlr2(-/-) littermates were used to reconstitute lethally irradiated Ldlr(-/-) mice. Following a recovery period, mice were placed either on a diet containing 21% saturated fat - 0.15% cholesterol for 8 weeks to study early-stage atherosclerosis, or on a diet richer in cholesterol (1.5%) for 16 weeks to study late-stage atherosclerosis. Donor cell Tlr2 genotype did not alter serum cholesterol levels or lipoprotein profiles in recipient animals. After 8 weeks on the 0.15% cholesterol diet, deficiency of TLR-2 expression on cells of BM origin reduced atherosclerosis in the aortic root and the aortic arch in both genders of mice. In contrast, the BM recipients who received the 1.5% cholesterol diet for 16 weeks showed much larger lesions in the aortic root, and TLR-2 deficiency in BM cells failed to provide protection. Thus, TLR-2 expression in BM-derived cells contributes primarily to early stage atherosclerosis.

Deficiency of invariant V alpha 14 natural killer T cells decreases atherosclerosis in LDL receptor null mice.

AIMS: CD1d-restricted natural killer T (NKT) cells function by regulating numerous immune responses during innate and adaptive immunity. Depletion of all populations of CD1d-dependent NKT cells has been shown by several groups to reduce atherosclerosis in two different mouse models of the disease. In this study, we determined if removal of a single (V alpha 14) NKT cell population protects mice from the disease. METHODS AND RESULTS: Targeted deletion of the J alpha 18 gene results in selective depletion of CD1d-dependent V alpha 14 NKT cells in C57BL/6 mice without affecting the population of other NKT, NK, and conventional T cells. Therefore, to study the effect of V alpha 14 NKT cell depletion on the progression of atherosclerosis, we examined the extent of lesion formation using paired littermate LDL receptor null mice that were either +/+ or -/- for the J alpha 18 gene following the feeding of these mice a cholesterol- and fat-enriched diet for 8 weeks. At the end of the study, we found no difference in either serum total- or lipoprotein-cholesterol distributions between groups. However, quantification of atherosclerosis revealed that V alpha 14 NKT cell deficiency significantly decreased lesion size in the aortic root (20-28%) and arch (28-38%) in both genders of mice. By coupling the techniques of laser capture microdissection with quantitative real-time RT-PCR, we found that expression of the proatherogenic cytokine interferon (IFN)-gamma was significantly reduced in lesions from J alpha 18-/- mice. CONCLUSION: This study is the first to identify a specific subpopulation of NKT cells that promotes atherosclerosis via a mechanism appearing to involve IFN-gamma expression.

Cholesteryl ester transfer protein (CETP) expression protects against diet induced atherosclerosis in SR-BI deficient mice.

OBJECTIVE: To determine whether expression of the human CETP transgene protects against diet-induced atherosclerosis in SR-BI deficient mice. METHODS AND RESULTS: SR-BI deficient (-/-) mice were crossed with CETP transgenic (CETPtg) mice to produce a colony of SR-BI(-/-) x CETPtg mice in a C57Bl/6 background. Age and sex matched groups of genetically modified and wild-type C57Bl/6 mice were fed a high fat, high cholesterol diet for 22 weeks. In both wild-type and SR-BI(-/-) mice, expression of the CETP transgene reduced the cholesterol content and increased the density of lipoprotein particles in the HDL density range. In SR-BI(-/-) x CETPtg mice, CETP activity inversely correlated with total plasma cholesterol levels and shifted the buoyant HDL typical of SR-BI deficiency toward a more normal density HDL particle. Atherosclerosis at the level of the aortic arch was evident in both male and female SR-BI deficient mice but occurred to a greater extent in the females. Expression of CETP markedly attenuated the development of atherosclerosis in SR-BI deficient mice fed an atherogenic diet (P<0.003). CONCLUSIONS: Expression of the human CETP transgene protects SR-BI deficient mice from atherosclerosis, consistent with a role for CETP in remodeling HDL and providing an alternative pathway for the selective uptake of HDL-CE by the liver.

Generation of CD133+ cells from CD133- peripheral blood mononuclear cells and their properties.

OBJECTIVE: CD133 may be the most specific marker of endothelial progenitor cells (EPCs), which are thought to be largely confined to the bone marrow milieu. This study reports on the phenotypic characterization and functional analysis of human CD133+ cells and their generation from cells in the peripheral circulation. METHODS: Adult human CD133+ and CD133- cells were isolated from peripheral blood mononuclear cells, and the generation of CD133+ cells in culture was attempted using different culture combinations. The phenotypic, migratory, adhesive, and angiogenic properties of the native and generated populations were investigated. RESULTS: In adherent and in suspension culture systems, CD133+ cells also expressing CD34 and VEGFR-2 were successfully derived from a previously CD133- population. The migratory potential of CD133+ cells was enhanced by the presence of the CD133- cells. Also, the CD133+ cells derived from the CD133- cells demonstrated improved adhesion to extracellular matrix and endothelial monolayer substrates, and their contribution to in vitro angiogenesis was enhanced compared to freshly isolated CD133+ cells. CONCLUSIONS: These results demonstrate a source of blood CD133+ cells other than direct mobilization from the bone marrow. Cellular interaction was observed between fractions, with CD133+ cells showing better in vitro function in the presence of CD133- cells. These findings provide a novel source for CD133+ cells and a rationale for the investigation of angiogenic cell recruitment or delivery strategies involving more than one cell type at ischemic sites.

Interleukin-18 enhances atherosclerosis in apolipoprotein E(-/-) mice through release of interferon-gamma.

We have previously shown that interferon-gamma (IFN-gamma) is a potent enhancer of atherogenesis. Interleukin-18 (IL-18) promotes inflammatory responses through release of IFN-gamma, although it can also exert direct actions on other inflammatory mediators. In this present study, we determined the effects of IL-18 on atherogenesis and the role of IFN-gamma in this response. Male apolipoprotein E(-/-) mice (apoE(-/-); aged 16 weeks, n=10/group) were fed a normal diet and injected intraperitoneally for 30 days with either recombinant IL-18 (30 ng/g/day) or saline. Atherosclerotic lesion size was quantified in 2 vascular beds: the ascending aorta and the aortic arch. IL-18 administration did not affect serum cholesterol concentrations or lipoprotein-cholesterol distribution; however, exogenous IL-18 administration increased lesion size 2-fold in both the ascending aorta (50 642 +/- 12 515 versus 112 399 +/- 13 227 microm(2) P=0.004; saline versus IL-18 groups, respectively) and the aortic arch (3.1 +/- 0.3% versus 6.2 +/- 0.9% area, P=0.006). Exogenous IL-18 promoted a 4-fold increase in the number of lesion-associated T lymphocytes (11 +/- 3 versus 50 +/- 5 cells; P<0.0001) and cells expressing major histocompatability complex class II (9 +/- 3 versus 40 +/- 6 cells; P=0.0002). To determine the role of IFN-gamma production in this response, exogenous IL-18 was administered to apoE(-/-) mice that were IFN-gamma deficient. These studies demonstrated that lack of endogenous IFN-gamma ablated the effects of IL-18 on atherosclerosis. Therefore, these data strongly implicates IL-18 in the atherogenic process and suggests that IL-18 increases lesion development through enhancement of an inflammatory response involving an IFN-gamma-dependent mechanism. The full text of this article is available at http://www.circresaha.org.

Correction: Low-Dose Irradiation Affects Expression of Inflammatory Markers in the Heart of ApoE -/- Mice.

[This corrects the article DOI: 10.1371/journal.pone.0119661.].

Novel antiinflammatory vascular benefits of systemic and stent-based delivery of ethylisopropylamiloride.

BACKGROUND: Recently, we demonstrated that the amiloride derivative ethylisopropylamiloride (EIPA) limits vascular smooth muscle cell growth and migration. The purpose of the present experiments was to determine whether EIPA can also reduce the inflammatory component of atherogenesis and stent neointima formation. METHODS AND RESULTS: To determine the effect of EIPA on the early inflammatory stages of atherogenesis, apolipoprotein E null mice (apoE-/-) fed an atherogenic diet received a subcutaneous pump infusion of either EIPA (3 mg x kg(-1)d(-1)) or the control vehicle for 4 weeks. The en face aortic area of atherosclerotic lesions and the subendothelial accumulation of macrophages were reduced by 46% and 38%, respectively, in EIPA-treated mice. Moreover, the number of vascular cell adhesion molecule-1 (VCAM-1) immunopositive lumenal endothelial cells was 59% less in the EIPA treatment group. In vitro, there was a concentration-dependent inhibition of lipopolysaccharide (LPS)-induced VCAM-1 expression with a corresponding 37% reduction in U-937 cell adhesion to endothelial cells. EIPA also reduced LPS-stimulated nuclear factor-kappaB (NF-kappaB) activation as reflected by a 66% reduction in NF-kappaB nuclear translocation. Finally, to test the effect of EIPA on the early inflammatory reaction to stent implantation, stents coated with jelly alone or jelly plus EIPA were implanted into rabbit iliac arteries. Four weeks later, the stent neointimal area, abundance of peristrut macrophages, and density of intimal smooth muscle cells were reduced by 38%, 47%, and 37%, respectively, for EIPA stents. CONCLUSIONS: EIPA downregulates endothelial cell activation of NF-kappaB and VCAM-1 expression and attenuates the early inflammatory stages of atherogenesis and stent intimal formation.

Nobiletin, a citrus flavonoid isolated from tangerines, selectively inhibits class A scavenger receptor-mediated metabolism of acetylated LDL by mouse macrophages.

Flavonoids are a class of chemically related polyphenols that are nearly ubiquitous in nature. Of the more-than 4000 flavonoids thus identified, citrus fruit-derived flavonoids are suggested to have an inverse association with the occurrence of coronary heart disease via their ability to reduce plasma cholesterol concentrations. Our current studies examined whether citrus flavonoids possess an additional antiatherogenic effect by modulating macrophage metabolism of the specific class A scavenger receptor (SR-A) ligand, acetylated LDL (acLDL). In this study, both acLDL-metabolism and SR-A expression by cultured murine J774A.1 macrophages was examined following 24 h pretreatment (100 microM) with the flavonoids: naringenin (from grapefruit), hesperetin (from oranges), and tangeretin and nobiletin (from tangerines). Of these flavonoids, only nobiletin inhibited (50-72%) acLDL metabolism as measured by both cellular cholesterol ester mass and [3H]oleate incorporation into cholesterol esters. This nobiletin-mediated effect was specific for SR-A and not a global effect on lipoprotein metabolism by the macrophage, as all four citrus flavonoids significantly reduce the metabolism of beta-VLDL, which is primarily taken up by macrophages via the LDL receptor. Nevertheless, nobiletin did not affect SR-A protein expression, as measured by Western blot analysis, nor was cell surface expression of SR-A affected as measured by 4 degrees C binding studies using [125I]acLDL. In conclusion, our findings suggest that in addition to reducing plasma cholesterol concentrations, nobiletin may prevent atherosclerosis at the level of the vascular wall by inhibiting macrophage foam-cell formation.

Depletion of natural killer cell function decreases atherosclerosis in low-density lipoprotein receptor null mice.

OBJECTIVE: Natural killer (NK) cells are a key component of innate immunity. Despite being identified in human and mouse atherosclerotic lesions, the role of NK cells in the disease process in unknown. To determine this role, we created chimeric atherosclerosis-susceptible low-density lipoprotein (LDL) receptor null (ldl-r-/-) mice that were deficient in functional NK cells through expression of a transgene encoding for Ly49A. METHODS AND RESULTS: Bone marrow cells from Ly49A transgenic and nontransgenic littermates were used to repopulate the hematopoietic system of lethally-irradiated female ldl-r-/- mice. After a recovery period to permit sufficient engraftment, mice were placed on a diet enriched in saturated fat and cholesterol. After 8 weeks, there was no difference in either serum total cholesterol concentrations or lipoprotein cholesterol distribution in mice repopulated with nontransgenic versus Ly49A transgenic marrow cells. Using immunohistochemistry, we detected NK cells in atherosclerotic lesions of both groups of mice. However, deficiency of functional NK cells significantly reduced the size of atherosclerosis by 70% (P=0.0002) in cross-sectional analysis of the aortic root and by 38% (P=0.004) in en face analysis of the intimal surface of the aortic arch. CONCLUSIONS: These studies demonstrate that NK cells infiltrate the vessel wall and promote atherosclerotic lesion development.

Low-dose irradiation affects expression of inflammatory markers in the heart of ApoE -/- mice.

Epidemiological studies indicate long-term risks of ionizing radiation on the heart, even at moderate doses. In this study, we investigated the inflammatory, thrombotic and fibrotic late responses of the heart after low-dose irradiation (IR) with specific emphasize on the dose rate. Hypercholesterolemic ApoE-deficient mice were sacrificed 3 and 6 months after total body irradiation (TBI) with 0.025, 0.05, 0.1, 0.5 or 2 Gy at low (1 mGy/min) or high dose rate (150 mGy/min). The expression of inflammatory and thrombotic markers was quantified in frozen heart sections (CD31, E-selectin, thrombomodulin, ICAM-1, VCAM-1, collagen IV, Thy-1, and CD45) and in plasma samples (IL6, KC, MCP-1, TNFα, INFγ, IL-1ß, TGFß, INFγ, IL-10, sICAM-1, sE-selectin, sVCAM-1 and fibrinogen) by fluorescence analysis and ELISA. We found that even very low irradiation doses induced adaptive late responses, such as increases of capillary density and changes in collagen IV and Thy-1 levels indicating compensatory regulation. Slight decreases of ICAM-1 levels and reduction of Thy 1 expression at 0.025-0.5 Gy indicate anti-inflammatory effects, whereas at the highest dose (2 Gy) increased VCAM-1 levels on the endocardium may represent a switch to a pro-inflammatory response. Plasma samples partially confirmed this pattern, showing a decrease of proinflammatory markers (sVCAM, sICAM) at 0.025-2.0 Gy. In contrast, an enhancement of MCP-1, TNFα and fibrinogen at 0.05-2.0 Gy indicated a proinflammatory and prothrombotic systemic response. Multivariate analysis also revealed significant age-dependent increases (KC, MCP-1, fibrinogen) and decreases (sICAM, sVCAM, sE-selectin) of plasma markers. This paper represents local and systemic effects of low-dose irradiation, including also age- and dose rate-dependent responses in the ApoE-/- mouse model. These insights in the multiple inflammatory/thrombotic effects caused by low-dose irradiation might facilitate an individual evaluation and intervention of radiation related, long-term side effects but also give important implications for low dose anti-inflammatory radiotherapy.

IFN-gamma deficiency exerts gender-specific effects on atherogenesis in apolipoprotein E-/- mice.

We have shown recently that administration of exogenous interferon-gamma (IFN-gamma) to apolipoprotein E (apoE)(-/-) mice augmented atherogenesis. In the present study, we examined whether deficiency of endogenous IFN-gamma would reduce atherosclerosis in apoE(-/-) mice. Compound-deficient mice were generated by crossing strain-matched IFN-gamma(-/-) and apoE(-/-) mice and comparing them to apoE(-/-) mice. Groups of both genders were fed either a normal or a high-fat diet. IFN-gamma deficiency did not affect serum cholesterol concentrations or lipoprotein-cholesterol distributions in any groups. IFN-gamma deficiency had no effect on serum triglyceride concentrations, except for an increase noted in males fed a normal diet. The extent of atherosclerosis was determined in tissue sections of the ascending aorta and on the surface of the aortic arch. During feeding of normal diets, IFN-gamma deficiency had no effect on the extent of atherosclerosis in female mice in either vascular bed. In contrast, in male mice fed normal diet, IFN-gamma deficiency markedly decreased lesion size in both vascular beds. During feeding of high-fat diets, IFN-gamma deficiency also had no effect on lesion size in females but profoundly decreased lesion size in the aortic root of male mice. IFN-gamma deficiency had no effect on the abundance of T lymphocytes or MHC class II-positive cells in aortic root lesions of females. By comparison, both these parameters were reduced in lesions of male mice. Therefore, IFN-gamma deficiency decreased atherogenesis, potentially by decreasing T lymphocyte presence and cell activation, without influencing serum cholesterol concentrations. However, this effect is strikingly restricted to male mice.

Cardiovascular changes in atherosclerotic ApoE-deficient mice exposed to Co60 (γ) radiation.

BACKGROUND: There is evidence for a role of ionizing radiation in cardiovascular diseases. The goal of this work was to identify changes in oxidative and nitrative stress pathways and the status of the endothelinergic system during progression of atherosclerosis in ApoE-deficient mice after single and repeated exposure to ionizing radiation. METHODS AND RESULTS: B6.129P2-ApoE tmlUnc mice on a low-fat diet were acutely exposed (whole body) to Co60 (γ) (single dose 0, 0.5, and 2 Gy) at a dose rate of 36.32 cGy/min, or repeatedly (cumulative dose 0 and 2 Gy) at a dose-rate of 0.1 cGy/min for 5 d/wk, over a period of 4 weeks. Biological endpoints were investigated after 3-6 months of recovery post-radiation. The nitrative stress marker 3-nitrotyrosine and the vasoregulator peptides endothelin-1 and endothelin-3 in plasma were increased (p<0.05) in a dose-dependent manner 3-6 months after acute or chronic exposure to radiation. The oxidative stress marker 8-isoprostane was not affected by radiation, while plasma 8-hydroxydeoxyguanosine and L-3,4-dihydroxyphenylalanine decreased (p<0.05) after treatment. At 2Gy radiation dose, serum cholesterol was increased (p = 0.008) relative to controls. Percent lesion area increased (p = 0.005) with age of animal, but not with radiation treatment. CONCLUSIONS: Our observations are consistent with persistent nitrative stress and activation of the endothelinergic system in ApoE-/- mice after low-level ionizing radiation exposures. These mechanisms are known factors in the progression of atherosclerosis and other cardiovascular diseases.

Loss of cellular inhibitor of apoptosis protein 2 reduces atherosclerosis in atherogenic apoE-/- C57BL/6 mice on high-fat diet.

BACKGROUND: Cellular inhibitor of apoptosis protein 2 (cIAP2) is predicted to participate in atherosclerosis; however, its direct role in atherosclerosis development has not been investigated. We aimed to examine and assess the loss of cIAP2 on atherosclerosis lesion development. METHODS AND RESULTS: We used apoE-/- C57BL/6 male mice, either cIAP2-/- or cIAP2+/+. At 8 weeks, mice were fed a high-fat diet (HFD) for 4 and 12 weeks. Aortic root was serially sectioned and stained with Sudan IV, CD68, α-actin, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). cIAP2-/- mice displayed a significant decrease in atherosclerotic lesion's macrophage number after 4 weeks of HFD. Similarly, decrease in lesion area at 4 and 12 weeks HFD was detected by use of en face analysis (cIAP2-/- 0.58 ± 0.37% versus cIAP2+/+ 1.51 ± 0.79% [P = 0.0056]); (cIAP2-/- 9.34 ± 4.88% versus cIAP2+/+ 17.65 ± 6.24% [P = 0.0019]). Aortic root lesion area after 4 and 12 weeks of HFD also decreased (cIAP2-/- 0.0328 ± 0.014 mm2 versus cIAP2+/+ 0.0515 ± 0.021 mm2 [P = 0.022]); (cIAP2-/- 0.3614 ± 0.1157 mm2 versus cIAP2+/+ 0.4901 ± 0.125 mm2 [P = 0.065]). TUNEL analysis after 4 and 12 weeks of HFD showed a 2.5-fold increase in TUNEL+ cells (cIAP2-/- 4.47 ± 2.26% versus cIAP2+/+ 1.74 ± 0.98% [P = 0.036]); (cIAP2-/- 2.39 ± 0.75% versus cIAP2+/+ 1.29 ± 0.47% [P = 0.032]). Smooth muscle cell content in cIAP2-/- mice was 3.075 ± 3.3% compared with cIAP2+/+ with 0.085 ± 0.1% (P = 0.0071). CONCLUSIONS: Results uncover a key role for cIAP2 in atherosclerotic lesion development, and targeting it may represent a novel therapeutic strategy.

Insulin-degrading enzyme deficiency in bone marrow cells increases atherosclerosis in LDL receptor-deficient mice.

BACKGROUND: Insulin-degrading enzyme (IDE), a protease implicated in several chronic diseases, associates with the cytoplasmic domain of the macrophage Type A scavenger receptor (SR-A). Our goal was to investigate the effect of IDE deficiency (Ide(-/-)) on diet-induced atherosclerosis in low density lipoprotein-deficient (Ldlr(-/-)) mice and on SR-A function. METHODS: Irradiated Ldlr(-/-) or Ide(-/-)Ldlr(-/-) mice were reconstituted with wild-type or Ide(-/-) bone marrow and, 6 weeks later, were placed on a high-fat diet for 8 weeks. RESULTS: After 8 weeks on a high-fat diet, male Ldlr(-/-) recipients of Ide(-/-) bone marrow had more atherosclerosis, higher serum cholesterol and increased lesion-associated ß-amyloid, an IDE substrate, and receptor for advanced glycation end products (RAGE), a proinflammatory receptor for ß-amyloid, compared to male Ldlr(-/-) recipients of wild-type bone marrow. IDE deficiency in male Ldlr(-/-) recipient mice did not affect atherosclerosis or cholesterol levels and moderated the effects of IDE deficiency of bone marrow-derived cells. No differences were seen between Ldlr(-/-) and Ide(-/-)Ldlr(-/-) female mice reconstituted with Ide(-/-) or wild-type bone marrow. IDE deficiency in macrophages did not alter SR-A levels, cell surface SR-A, or foam cell formation. CONCLUSION: IDE deficiency in bone marrow-derived cells results in larger atherosclerotic lesions, increased lesion-associated Aß and RAGE, and higher serum cholesterol in male, Ldlr(-/-) mice.

Caspase-1 deficiency decreases atherosclerosis in apolipoprotein E-null mice.

BACKGROUND: Caspase-1 is a cysteine protease that contributes to mammalian immunity through proteolytic activation of the proinflammatory cytokines, interleukin (IL)-1ß and IL-18. METHODS: To determine if caspase-1 deficiency can protect apolipoprotein E-null (Apoe(-/-)) mice from atherosclerosis, gender-matched, paired-littermate Apoe(-/-) mice with (Casp1(+/+)Apoe(-/-)) or without (Casp1(-/-)Apoe(-/-)) a functional caspase-1 (Casp1) gene were fed either a low fat diet for 26 weeks, or a saturated fat and cholesterol-enriched diet for 8 weeks. Plasma lipids and lipoproteins were determined and atherosclerosis was quantified in the aortic sinus and aortic arch. RESULTS: On either diet, caspase-1 deficiency did not affect total serum cholesterol concentrations and lipoprotein-cholesterol distributions. However, caspase-1 deficiency significantly decreased atherosclerosis in the ascending aorta by 35%-45% in both sexes of mice fed either diet. We further examined atherosclerotic lesions for 2 indices of immune cell activation: Major Histocompatibility Complex (MHC) class II and interferon (IFN)-γ expression. There was a 40%-50% reduction in the number of lesion-associated cells expressing MHC class II from both sexes of Casp1(-/-)Apoe(-/-) mice compared with Casp1(+/+)Apoe(-/-) mice and, a significant reduction in lesion-associated IFN-γ in female Casp1(-/-)Apoe(-/-) compared with their Casp1(+/+)Apoe(-/-) counterparts. CONCLUSIONS: We conclude that caspase-1 promotes atherosclerosis by enhancing the inflammatory status of the lesion through a mechanism likely involving activation of lesion-associated immune cells and IFN-γ expression.