Yan Ru Su

Reduced Macrophage Apoptosis Is Associated With Accelerated Atherosclerosis in Low-Density Lipoprotein Receptor-Null Mice

Objective— The majority of apoptotic cells in atherosclerotic lesions are macrophages. However, the pathogenic role of macrophage apoptosis in the development of atherosclerosis remains unclear. Elevated expression of Bax, one of the pivotal proapoptotic proteins of the Bcl-2 family, has been found in human atherosclerotic plaques. Activation of Bax also occurs in free cholesterol–loaded and oxysterol-treated mouse macrophages. In this study, we examined the effect of Bax deficiency in bone marrow–derived leukocytes on the development of atherosclerosis in low-density lipoprotein receptor–null (LDLR−/−) mice. Methods and Results— Fourteen 8-week-old male LDLR−/− mice were lethally irradiated and reconstituted with either wild-type (WT) C57BL6 or Bax-null (Bax−/−) bone marrow. Three weeks later, the mice were challenged with a Western diet for 10 weeks. No differences were found in the plasma cholesterol level between the WT and Bax−/− group. However, quantitation of cross sections from proximal aorta revealed a 49.2% increase (P=0.0259) in the mean lesion area of the Bax−/− group compared with the WT group. A 53% decrease in apoptotic macrophages in the Bax−/− group was found by TUNEL staining (P<0.05). Conclusions— The reduction of apoptotic activity in macrophages stimulates atherosclerosis in LDLR−/− mice, which is consistent with the hypothesis that macrophage apoptosis suppresses the development of atherosclerosis.

Inactivation of Macrophage Scavenger Receptor Class B Type I Promotes Atherosclerotic Lesion Development in Apolipoprotein E–Deficient Mice

Background—Scavenger receptor class B type I (SR-BI) is expressed in macrophages, where it has been proposed to facilitate cholesterol efflux. However, direct evidence that the expression of macrophage SR-BI is protective against atherosclerosis is lacking. In this study, we examined the in vivo role of macrophage SR-BI in atherosclerotic lesion development in the apolipoprotein (apo) E–deficient mouse model. Methods and Results—ApoE-deficient mice with (n=16) or without (n=15) expression of macrophage SR-BI were created by transplanting lethally irradiated apoE-deficient mice with bone marrow cells collected from SR-BI−/− apoE−/− mice or SR-BI+/+ apoE−/− mice. The recipient mice were fed a chow diet for 12 weeks after transplantation for analysis of atherosclerosis. Quantification of macrophage SR-BI mRNA by real-time reverse transcription–polymerase chain reaction indicated successful engraftment of donor bone marrow and inactivation of macrophage SR-BI in recipient mice reconstituted with SR-BI−/− apoE−/− bone marrow. There were no significant differences in plasma lipid levels, lipoprotein distributions, and HDL subpopulations between the 2 groups. Analysis of the proximal aorta demonstrated an 86% increase in mean atherosclerotic lesion area in SR-BI−/− apoE−/− → apoE−/− mice compared with SR-BI+/+ apoE−/− → apoE−/− mice (109.50±18.08 versus 58.75±9.58×103 &mgr;m2; mean±SEM, P =0.017). No difference in cholesterol efflux from SR-BI+/+ apoE−/− or SR-BI−/− apoE−/− macrophages to HDL or apoA-I discs was detected. Conclusions—Expression of macrophage SR-BI protects mice against atherosclerotic lesion development in apoE-deficient mice in vivo without influencing plasma lipids, HDL subpopulations, or cholesterol efflux. Thus, macrophage SR-BI plays an antiatherogenic role in vivo, providing a new therapeutic target for the design of strategies to prevent and treat atherosclerosis.

Overexpression of Human Apolipoprotein A-I Preserves Cognitive Function and Attenuates Neuroinflammation and Cerebral Amyloid Angiopathy in a Mouse Model of Alzheimer Disease

To date there is no effective therapy for Alzheimer disease (AD). High levels of circulating high density lipoprotein (HDL) and its main protein, apolipoprotein A-I (apoA-I), reduce the risk of cardiovascular disease. Clinical studies show that plasma HDL cholesterol and apoA-I levels are low in patients with AD. To investigate if increasing plasma apoA-I/HDL levels ameliorates AD-like memory deficits and amyloid-β (Aβ) deposition, we generated a line of triple transgenic (Tg) mice overexpressing mutant forms of amyloid-β precursor protein (APP) and presenilin 1 (PS1) as well as human apoA-I (AI). Here we show that APP/PS1/AI triple Tg mice have a 2-fold increase of plasma HDL cholesterol levels. When tested in the Morris water maze for spatial orientation abilities, whereas APP/PS1 mice develop age-related learning and memory deficits, APP/PS1/AI mice continue to perform normally during aging. Interestingly, no significant differences were found in the total level and deposition of Aβ in the brains of APP/PS1 and APP/PS1/AI mice, but cerebral amyloid angiopathy was reduced in APP/PS1/AI mice. Also, consistent with the anti-inflammatory properties of apoA-I/HDL, glial activation was reduced in the brain of APP/PS1/AI mice. In addition, Aβ-induced production of proinflammatory chemokines/cytokines was decreased in mouse organotypic hippocampal slice cultures expressing human apoA-I. Therefore, we conclude that overexpression of human apoA-I in the circulation prevents learning and memory deficits in APP/PS1 mice, partly by attenuating neuroinflammation and cerebral amyloid angiopathy. These findings suggest that elevating plasma apoA-I/HDL levels may be an effective approach to preserve cognitive function in patients with AD.

Quantitative and Qualitative Differences in Proatherogenic NKT Cells in Apolipoprotein E–Deficient Mice

Background—Atherosclerosis is a disease marked by lipid accumulation and inflammation. Recently, atherosclerosis has gained recognition as an autoimmune-type syndrome characterized by increased activation of the innate and acquired immune systems. Natural killer T (NKT) cells have characteristics of both conventional T cells and NK cells and recognize glycolipid antigens presented in association with CD1d molecules on antigen-presenting cells. The capacity of NKT cells to respond to lipid antigens and modulate innate and acquired immunity suggests that they may play a role in atherogenesis. Methods and Results—We examined the role of NKT cells in atherogenesis and how the atherosclerotic environment affects the NKT cell population itself. The data show that CD1d-deficiency in male apolipoprotein E–deficient (apoE0) mice results in reduction in atherosclerosis, and treatment of apoE0 mice with α-galactosylceramide, a potent and specific NKT cell activator, results in a 2-fold increase in atherosclerosis. Interestingly, we demonstrate that α-galactosylceramide–induced interferon-γ responses and numbers of NKT cells in apoE0 mice show age-dependent qualitative and quantitative differences as compared with age-matched wild-type mice. Conclusions—Collectively, these findings reveal that hyperlipidemia and atherosclerosis have significant effects on NKT cell responses and that these cells are proatherogenic.

Reduced ABCA1-Mediated Cholesterol Efflux and Accelerated Atherosclerosis in Apolipoprotein E–Deficient Mice Lacking Macrophage-Derived ACAT1

Background—Macrophage acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) and apolipoprotein E (apoE) have been implicated in regulating cellular cholesterol homeostasis and therefore play critical roles in foam cell formation. Deletion of either ACAT1 or apoE results in increased atherosclerosis in hyperlipidemic mice, possibly as a consequence of altered cholesterol processing. We have studied the effect of macrophage ACAT1 deletion on atherogenesis in apoE-deficient (apoE−/−) mice with or without the restoration of macrophage apoE. Methods and Results—We used bone marrow transplantation to generate apoE−/− mice with macrophages of 4 genotypes: apoE+/+/ACAT1+/+ (wild type), apoE+/+/ACAT1−/− (ACAT−/−), apoE−/−/ACAT1+/+ (apoE−/−), and apoE−/−/ACAT1−/− (2KO). When macrophage apoE was present, plasma cholesterol levels normalized, and ACAT1 deficiency did not have significant effects on atherogenesis. However, when macrophage apoE was absent, ACAT1 deficiency increased atherosclerosis and apoptosis in the proximal aorta. Cholesterol efflux to apoA-I was significantly reduced (30% to 40%; P<0.001) in ACAT1−/− peritoneal macrophages compared with ACAT1+/+ controls regardless of apoE expression. 2KO macrophages had a 3- to 4-fold increase in ABCA1 message levels but decreased ABCA1 protein levels relative to ACAT1+/+ macrophages. Microarray analyses of ACAT1−/− macrophages showed increases in proinflammatory and procollagen genes and decreases in genes regulating membrane integrity, protein biosynthesis, and apoptosis. Conclusions—Deficiency of macrophage ACAT1 accelerates atherosclerosis in hypercholesterolemic apoE−/− mice but has no effect when the hypercholesterolemia is corrected by macrophage apoE expression. However, ACAT1 deletion impairs ABCA1-mediated cholesterol efflux in macrophages regardless of apoE expression. Changes in membrane stability, susceptibility to apoptosis, and inflammatory response may also be important in this process.

Genetic Markers at the Leptin (OB) Locus Are Not Significantly Linked to Hypertension in African Americans

Increased body mass index (BMI) has been correlated with increased blood pressure in human populations. To examine the role of the leptin gene (OB) in essential hypertension in African Americans, we performed affected sib pair analysis on a set of 103 hypertensive African American sibships using four highly polymorphic markers at the human leptin locus. No evidence of linkage was detected between these markers and the phenotype of essential hypertension either in these sibships or in a severely obese subset of 46 sibships in which each sibling had a BMI > or = 85th percentile for the US population. Using BMI rather than hypertension as a quantitative trait, we found significant linkage for the marker D7S504 (P=0.029) but not for the other markers. Significance strengthened in the overweight subset of sibships for this marker (P=0.001), and there was a trend of lower P values for the other three markers. However, multipoint analysis with the use of all four markers simultaneously to estimate linkage between BMI and the leptin locus did not demonstrate a statistically significant relationship. Analysis of the coding region of the leptin gene (exons 2 and 3) by single-strand conformational polymorphism revealed a rare Ile-Val polymorphism at amino acid 45 but revealed no other alterations. These results suggest that the OB gene is not a major contributor to the phenotype of essential hypertension in African Americans, although a minor contribution to the phenotype of extreme obesity in this group cannot be ruled out.

Cadherin-11 Regulates Cell–Cell Tension Necessary for Calcific Nodule Formation by Valvular Myofibroblasts

Objective—Dystrophic calcific nodule formation in vitro involves differentiation of aortic valve interstitial cells (AVICs) into a myofibroblast phenotype. Interestingly, inhibition of the kinase MAPK Erk kinase (MEK)1/2 prevents calcific nodule formation despite leading to myofibroblast activation of AVICs, indicating the presence of an additional mechanotransductive component required for calcific nodule morphogenesis. In this study, we assess the role of transforming growth factor &bgr;1–induced cadherin-11 expression in calcific nodule formation. Methods and Results—As shown previously, porcine AVICs treated with transforming growth factor &bgr;1 before cyclic strain exhibit increased myofibroblast activation and significant calcific nodule formation. In addition to an increase in contractile myofibroblast markers, transforming growth factor &bgr;1–treated AVICs exhibit significantly increased expression of cadherin-11. This expression is inhibited by the addition of U0126, a specific MEK1/2 inhibitor. The role of increased cadherin-11 is revealed through a wound assay, which demonstrates increased intercellular tension in transforming growth factor &bgr;1–treated AVICs possessing cadherin-11. Furthermore, when small interfering RNA is used to knockdown cadherin-11, calcific nodule formation is abrogated, indicating that robust cell–cell connections are necessary in generating tension for calcific nodule morphogenesis. Finally, we demonstrate enrichment of cadherin-11 in human calcified leaflets. Conclusion—These results indicate the necessity of cadherin-11 for dystrophic calcific nodule formation, which proceeds through an Erk1/2-dependent pathway.

ACAT1 Deficiency Disrupts Cholesterol Efflux and Alters Cellular Morphology in Macrophages

Objective— Acyl-coenzyme A: cholesterol acyltransferase (ACAT) converts intracellular free cholesterol (FC) into cholesteryl esters (CE) for storage in lipid droplets. Recent studies in our laboratory have shown that the deletion of the macrophage ACAT1 gene results in apoptosis and increased atherosclerotic lesion area in the aortas of hyperlipidemic mice. The objective of the current study was to elucidate the mechanism of the increased atherosclerosis. Methods and Results— CE storage and FC efflux were studied in ACAT1(−/−) peritoneal macrophages that were treated with acetylated low-density lipoprotein (acLDL). Our results show that efflux of cellular cholesterol was reduced by 25% in ACAT1-deficient cells compared with wild-type controls. This decrease occurred despite the upregulated expression of ABCA1, an important mediator of cholesterol efflux. In contrast, ACAT1 deficiency increased efflux of the cholesterol derived from acLDL by 32%. ACAT1-deficient macrophages also showed a 26% increase in the accumulation of FC derived from acLDL, which was associated with a 75% increase in the number of intracellular vesicles. Conclusions— Together, these data show that macrophage ACAT1 influences the efflux of both cellular and lipoprotein-derived cholesterol and propose a pathway for the pro-atherogenic transformation of ACAT1(−/−) macrophages.

Quantum dot mediated imaging of atherosclerosis

The progression of atherosclerosis is associated with leukocyte infiltration within lesions. We describe a technique for the ex vivo imaging of cellular recruitment in atherogenesis which utilizes quantum dots (QD) to color-code different cell types within lesion areas. Spectrally distinct QD were coated with the cell-penetrating peptide maurocalcine to fluorescently-label immunomagnetically isolated monocyte/macrophages and T lymphocytes. QD-maurocalcine bioconjugates labeled both cell types with a high efficiency, preserved cell viability, and did not perturb native leukocyte function in cytokine release and endothelial adhesion assays. QD-labeled monocyte/macrophages and T lymphocytes were reinfused in an ApoE(-/-) mouse model of atherosclerosis and age-matched controls and tracked for up to four weeks to investigate the incorporation of cells within aortic lesion areas, as determined by oil red O (ORO) and immunofluorescence ex vivo staining. QD-labeled cells were visible in atherosclerotic plaques within two days of injection, and the two cell types colocalized within areas of subsequent ORO staining. Our method for tracking leukocytes in lesions enables high signal-to-noise ratio imaging of multiple cell types and biomarkers simultaneously within the same specimen. It also has great utility in studies aimed at investigating the role of distinct circulating leukocyte subsets in plaque development and progression.

Whole exome sequencing identifies a causal RBM20 mutation in a large pedigree with familial dilated cardiomyopathy.

Background—Whole exome sequencing is a powerful technique for Mendelian disease gene discovery. However, variant prioritization remains a challenge. We applied whole exome sequencing to identify the causal variant in a large family with familial dilated cardiomyopathy of unknown pathogenesis. Methods and Results—A large family with autosomal dominant, familial dilated cardiomyopathy was identified. Exome capture and sequencing were performed in 3 remotely related, affected subjects predicted to share <0.1% of their genomes by descent. Shared variants were filtered for rarity, evolutionary conservation, and predicted functional significance, and remaining variants were filtered against 71 locally generated exomes. Variants were also prioritized using the Variant Annotation Analysis and Search Tool. Final candidates were validated by Sanger sequencing and tested for segregation. There were 664 shared heterozygous nonsense, missense, or splice site variants, of which 26 were rare (minor allele frequency ⩽0.001 or not reported) in 2 public databases. Filtering against internal exomes reduced the number of candidates to 2, and of these, a single variant (c.1907 G>A) in RBM20, segregated with disease status and was absent in unaffected internal reference exomes. Bioinformatic prioritization with Variant Annotation Analysis and Search Tool supported this result. Conclusions—Whole exome sequencing of remotely related dilated cardiomyopathy subjects from a large, multiplex family, followed by systematic filtering, identified a causal RBM20 mutation without the need for linkage analysis.