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Objective: Genome-wide studies showed that mutation in apoER2 (apolipoprotein E receptor-2) is additive with ε4 polymorphism in the APOE gene on cardiovascular disease risk in humans. ApoE or apoER2 deficiency also accelerates atherosclerosis lesion necrosis in hypercholesterolemic mice and promotes neointima formation after vascular injury. This study tests the hypothesis that apoE and apoER2 modulate vascular occlusive diseases through distinct mechanisms. Approach and Results: Carotid endothelial denudation induced robust neointima formation in both apoE −/− and apoER2-deficient Lrp8 −/− mice. The intima in apoE −/− mice was rich in smooth muscle cells, but the intima in Lrp8 −/− mice was cell-poor and rich in extracellular matrix. Vascular smooth muscle cells isolated from apoE −/− mice were hyperplastic whereas Lrp8 −/− smooth muscle cells showed reduced proliferation but responded robustly to TGF (transforming growth factor)-β–induced fibronectin synthesis indicative of a senescence-associated secretory phenotype, which was confirmed by increased β-galactosidase activity, p16 INK4a immunofluorescence, and number of multinucleated cells. Western blot analysis of cell cycle-associated proteins showed that apoER2 deficiency promotes cell cycle arrest at the metaphase/anaphase. Coimmunoprecipitation experiments revealed that apoER2 interacts with the catalytic subunit of protein phosphatase 2A. In the absence of apoER2, PP2A-C (protein phosphatase 2A catalytic subunit) failed to interact with CDC20 (cell-division cycle protein 20) thus resulting in inactive anaphase-promoting complex and impaired cell cycle exit. Conclusions: This study showed that apoER2 participates in APC (anaphase-promoting complex)/CDC20 complex formation during mitosis, and its absence impedes cytokinesis abscission thereby accelerating premature cell senescence and vascular disease. This mechanism is distinct from apoE deficiency, which causes smooth muscle cell hyperplasia to accelerate vascular disease.

Supplemental Digital Content is available in the text., Objective— Studies into the role of LRP1 (low-density lipoprotein receptor–related protein 1) in human lipid metabolism are scarce. Although it is known that a common variant in LRP1 (rs116133520) is significantly associated with HDL-C (high-density lipoprotein cholesterol), the mechanism underlying this observation is unclear. In this study, we set out to study the functional effects of 2 rare LRP1 variants identified in subjects with extremely low HDL-C levels. Approach and Results— In 2 subjects with HDL-C below the first percentile for age and sex and moderately elevated triglycerides, we identified 2 rare variants in LRP1: p.Val3244Ile and p.Glu3983Asp. Both variants decrease LRP1 expression and stability. We show in a series of translational experiments that these variants culminate in reduced trafficking of ABCA1 (ATP-binding cassette A1) to the cell membrane. This is accompanied by an increase in cell surface expression of SR-B1 (scavenger receptor class B type 1). Combined these effects may contribute to low HDL-C levels in our study subjects. Supporting these findings, we provide epidemiological evidence that rs116133520 is associated with apo (apolipoprotein) A1 but not with apoB levels. Conclusions— This study provides the first evidence that rare variants in LRP1 are associated with changes in human lipid metabolism. Specifically, this study shows that LRP1 may affect HDL metabolism by virtue of its effect on both ABCA1 and SR-B1.

Objective— Mice with adipocyte-specific inactivation of low-density lipoprotein receptor–related protein-1 (LRP1) are resistant to diet-induced obesity and hyperglycemia because of compensatory thermogenic response by muscle. However, the physiological function of LRP1 in mature adipocytes and its role in cardiovascular disease modulation are unknown. This study compared perivascular adipose tissues (PVAT) from wild-type ( adLrp1 +/+ ) and adipocyte-specific LRP1 knockout ( adLrp1 −/− ) mice in modulation of atherosclerosis progression. Approach and Results— Analysis of adipose tissues from adLrp1 +/+ and adLrp1 −/− mice after Western diet feeding for 16 weeks revealed that, in comparison to adLrp1 +/+ mice, the adipocytes in adLrp1 −/− mice were smaller, but their adipose tissues were more inflamed with increased monocyte–macrophage infiltration and inflammatory gene expression. The transplantation of PVAT from chow-fed adLrp1 +/+ and adLrp1 −/− mice into the area surrounding the carotid arteries of Ldlr −/− mice before feeding the Western diet revealed a contributory role of PVAT toward hypercholesterolemia-induced atherosclerosis. Importantly, recipients of adLrp1 −/− PVAT displayed a 3-fold increase in atherosclerosis compared with adLrp1 +/+ PVAT recipients. The increased atherosclerosis invoked by LRP1-deficient PVAT was associated with elevated monocyte–macrophage infiltration and inflammatory cytokine expression in the transplanted fat. Conclusions— PVAT provide outside-in signals through the adventitia to modulate atherosclerotic lesion progression in response to hypercholesterolemia. Moreover, adipocytes with LRP1 deficiency are dysfunctional and more inflamed. This latter observation adds the adipose tissue to the list of anatomic sites where LRP1 expression is important to protect against diet-induced atherosclerosis.

Introduction: Apolipoprotein E receptor 2 (ApoER2) is highly expressed in brain and testes, but also in macrophages, endothelial and vascular smooth muscle cells, as well as heart. Importantly, genome-wide association studies have linked polymorphisms in the LRP8 gene to familial and premature coronary artery disease (CAD) and myocardial infarction (MI). However, the mechanisms by which ApoER2, the protein encoded by the LRP8 gene, affect CAD and MI are incompletely understood. Hypothesis: The goal of the current study was to examine the function of ApoER2 in the heart. Methods and Results: Morphological analysis revealed an increase in heart size and cross-sectional area with elevated cardiomyocyte proliferation in mice deficient for ApoER2 compared to wild-type control mice. Echocardiography showed a significant increase in left ventricular diameter in ApoER2 KO mice, while no significant differences in fractional shortening and ejection fraction were observed. Loss of ApoER2 attenuated expression of Disabled-2 (Dab2) and Axin2, thereby preventing formation of the destruction complex, resulting in increased levels of β-catenin. Consistent with previous studies that demonstrated that stabilized β-catenin alters expression of the T-box protein Tbx20, ApoER2 deficiency was associated with increased Tbx20 expression, a transcription factor necessary and sufficient to promote cardiomyocyte proliferation. Conclusions: Together, these data indicate that ApoER2 promotes β-catenin degradation and modulates Tbx20 expression to restrain cardiomyocyte proliferation and heart size, providing a mechanism by which ApoER2 dysfunction may contribute to premature CAD and MI.

Cardiometabolic disease, emerging as a worldwide epidemic, is a combination of metabolic derangements leading to type 2 diabetes mellitus and cardiovascular disease. Genetic and environmental factors are linked through epigenetic mechanisms to the pathogenesis of cardiometabolic disease. Post-translational modifications of histone tails, including acetylation and deacetylation, epigenetically alter chromatin structure and dictate cell-specific gene expression patterns. The histone deacetylase family comprises 18 members that regulate gene expression by altering the acetylation status of nucleosomal histones and by functioning as nuclear transcriptional corepressors. Histone deacetylases regulate key aspects of metabolism, inflammation, and vascular function pertinent to cardiometabolic disease in a cell- and tissue-specific manner. Histone deacetylases also likely play a role in the metabolic memory of diabetes mellitus, an important clinical aspect of the disease. Understanding the molecular, cellular, and physiological functions of histone deacetylases in cardiometabolic disease is expected to provide insight into disease pathogenesis, risk factor control, and therapeutic development.

Objective— Perivascular adipose tissue (PVAT) expands during obesity, is highly inflamed, and correlates with coronary plaque burden and increased cardiovascular risk. We tested the hypothesis that PVAT contributes to the vascular response to wire injury and investigated the underlying mechanisms. Approach and Results— We transplanted thoracic aortic PVAT from donor mice fed a high-fat diet to the carotid arteries of recipient high-fat diet–fed low-density lipoprotein receptor knockout mice. Two weeks after transplantation, wire injury was performed, and animals were euthanized 2 weeks later. Immunohistochemistry was performed to quantify adventitial macrophage infiltration and neovascularization and neointimal lesion composition and size. Transplanted PVAT accelerated neointimal hyperplasia, adventitial macrophage infiltration, and adventitial angiogenesis. The majority of neointimal cells in PVAT-transplanted animals expressed α-smooth muscle actin, consistent with smooth muscle phenotype. Deletion of monocyte chemoattractant protein-1 in PVAT substantially attenuated the effects of fat transplantation on neointimal hyperplasia and adventitial angiogenesis, but not adventitial macrophage infiltration. Conditioned medium from perivascular adipocytes induced potent monocyte chemotaxis in vitro and angiogenic responses in cultured endothelial cells. Conclusions— These findings indicate that PVAT contributes to the vascular response to wire injury, in part through monocyte chemoattractant protein-1–dependent mechanisms.

Objective— Mitogen-activated protein kinase pathways play an important role in neointima formation secondary to vascular injury, in part by promoting proliferation of vascular smooth muscle cells (VSMC). Mixed-lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase that activates multiple mitogen-activated protein kinase pathways and has been implicated in regulating proliferation in several cell types. However, the role of MLK3 in VSMC proliferation and neointima formation is unknown. The aim of this study was to determine the function of MLK3 in the development of neointimal hyperplasia and to elucidate the underlying mechanisms. Approach and Results— Neointima formation was analyzed after endothelial denudation of carotid arteries from wild-type and MLK3-deficient mice. MLK3 deficiency promoted injury-induced neointima formation and increased proliferation of primary VSMC derived from aortas isolated from MLK3-deficient mice compared with wild-type mice. Furthermore, MLK3 deficiency increased the activation of p63Rho guanine nucleotide exchange factor, RhoA, and Rho kinase in VSMC, a pathway known to promote neointimal hyperplasia, and reconstitution of MLK3 expression attenuated Rho kinase activation. Furthermore, cJun NH 2 -terminal kinase activation was decreased in MLK3-deficient VSMC, and proliferation of wild-type but not MLK3 knockout cells treated with a cJun NH 2 -terminal kinase inhibitor was attenuated. Conclusions— We demonstrate that MLK3 limits RhoA activation and injury-induced neointima formation by binding to and inhibiting the activation of p63Rho guanine nucleotide exchange factor, a RhoA activator. In MLK3-deficient cells, activation of p63Rho guanine nucleotide exchange factor proceeds in an unchecked manner, leading to a net increase in RhoA pathway activation. Reconstitution of MLK3 expression restores MLK3/p63Rho guanine nucleotide exchange factor interaction, which is attenuated by feedback from activated cJun NH 2 -terminal kinase.

Abstract —Initial experiments revealed that low concentrations of apolipoprotein (apo) E (0.1 to 5 μg/mL) were effective in inhibiting platelet-derived growth factor (PDGF)–directed smooth muscle cell (SMC) migration by 60% to 80%. In contrast, higher concentrations of apoE, at 25 and 50 μg/mL, were necessary to achieve similar inhibition of PDGF-induced SMC proliferation. The potential role of nitric oxide (NO) in mediating the inhibitory effects of apoE was explored. Results showed that, although 0.1 to 5 μg/mL of apoE had no effect on NO production by SMCs, physiological concentrations of apoE (25 to 50 μg/mL) enhanced NO synthesis by 2-fold in a dose-dependent manner ( P N G -monomethyl- l -arginine ( P P

Atherosclerosis is a disease of large arteries that accounts for more than 50% of all deaths in the developed countries1 and, according to the World Health Organization prediction, it will gain a status of the major morbidity cause worldwide by the year 2010.2 Atherosclerosis is widely viewed as an inflammatory disease with hypercholesterolemia being a dominant underlying risk factor.3,4 It is believed to be initiated by retention of LDL particles in the lesion-prone areas, which is followed by monocyte recruitment and their differentiation into cholesterol-laden macrophage foam cells.1,5 Excessive cholesterol accumulation in macrophages exaggerates innate immune response that is manifested by upregulated production and secretion of inflammatory cytokines and chemokines, thus dramatically amplifying initial signal originated from the injured artery.6 See page 519 Despite the overall consensus on the causative roles of excessive cholesterol build-up and inflammation in the development and progression of atherosclerosis, the relationship between aberrant cholesterol metabolism and exaggerated innate immune response has not been totally established. Recent reports showing reduced inflammation in association with the cholesterol-lowering statin therapy provided indirect evidence linking cholesterol metabolism with regulation of immune response.7 However, this class of cholesterol lowering drugs not only inhibits cholesterol biosynthesis but also suppresses the synthesis of isoprenoids such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate. Thus, inhibition of prenylation pathways such as those required for activation of Ras and Rho GTPase family proteins may be responsible for some of the …

INTRODUCTION Apolipoprotein C-III (apoC-III), synthesized by the liver and intestine, is an inhibitor of LPL-mediated lipolysis and hepatic clearance of triglyceride-rich lipoproteins. ApoC-III overproduction is linked with hypertriglyceridemia and atherosclerosis. ApoC-III may also play an intracellular role in hepatic VLDL assembly/secretion. Little is known about the role of apoC-III in the intestine, although it is secreted on chylomicrons coincident with triglyceride absorption. HYPOTHESIS Given that overexpression of apoC-III results in high plasma triglyceride levels, we hypothesized that it might also stimulate intestinal triglyceride transport, thereby exacerbating plasma hypertriglyceridemia in human apoC-III transgenic (h-apoC-III tg) mice. METHODS 28-30 gram male h-apoC-III tg (on a C57BL/6J background) were fitted with both a mesenteric lymph cannula and a duodenal feeding tube, and received a continuous intraduodenal infusion of triglyceride (6 μmol of 3[H]-Triolein in 0.3ml of phosphate-buffered saline) for 6 hours with hourly lymph samples collected (n=11-12). At the end of the infusion period, luminal and mucosal contents and tissue samples were isolated. An advantage of this lymph fistula model is the ability to sample lymph continuously throughout the triglyceride infusion period while avoiding confounding effects of anesthesia and stomach emptying. RESULTS h-apoC-III tg mice had a decrease in lymph flow and a 43% reduction in lymphatic 3[H]-triglyceride transport compared to WT mice. The h-apoC-III tg mice had 10.0±2.3% of the total dose of 3[H]-triglyceride remaining in intestinal lumen; which was significantly higher than the 3.1±0.4% observed in WT mice. Thin layer chromatographic analysis of the luminal contents showed that h-apoC-III tg mice, as opposed to WT controls, had a significantly higher percentage of fatty acid. There were no significant differences in the luminal triglyceride, diglyceride, and monoglyceride composition between groups. CONCLUSION Our studies reveal a novel role for apoC-III in decreasing intestinal triglyceride transport distinct from its extracellular roles in plasma on lipoprotein lipase, and its intracellular role in hepatic VLDL synthesis and secretion.

During chronic overfeeding, adipose tissue expansion contributes to adipose inflammation, insulin resistance and obesity-related diabetes. Consequently, identifying mechanisms that promote “healthy” growth of adipose tissues could potentially improve obesity-related disease. We previously demonstrated that endogenous HDAC9 negatively regulates adipogenic differentiation in isolated preadipocytes. Here, we investigated the role of HDAC9 in regulating adipose tissue function and glucose intolerance in high fat fed mice. High fat feeding upregulated HDAC9 expression in adipose tissues, isolated preadipocytes and adipocytes, in conjunction with reduced expression of adipocyte differentiation-specific genes C/EBPα, PPARγ, FABP4, and adiponectin. HDAC9 gene knockout completely prevented these effects of high fat feeding on adipocyte differentiation-specific gene expression and adipogenic differentiation in vitro. HDAC9 knockout mice also gained less weight and exhibited less visceral adiposity, despite similar food intake, as compared to wild type mice during high fat feeding. In addition, HDAC9 gene deletion blunted elevations of plasma pro-inflammatory adipokines leptin and resistin, and resulted in improved systemic glucose tolerance, in the setting of high fat diet. Adipocytes were smaller in HDAC9 knockout mice, while ectopic lipid accumulation, as revealed by hepatosteatosis, following high fat feeding was completely prevented by HDAC9 gene knockout. Interestingly, adipose tissues of high fat fed HDAC9 knockout mice accumulated more anti-inflammatory M2 polarized macrophages and Treg cells, consistent with reduced adipose tissue inflammation. These findings suggest that HDAC9 disrupts adipose tissue function in high fat feeding, leading thereby to adipose tissue inflammation and glucose intolerance. Thus, HDAC9 could be a novel therapeutic target in the treatment of disorders associated with diet-induced obesity.

Inflammatory crosstalk between PV adipose tissue and the blood vessel wall has been proposed to contribute to the pathogenesis of atherosclerosis. We reported that PV adipocytes exhibit a pro-inflammatory phenotype, reduced state of differentiation, and altered expression of developmental genes as compared with subcutaneous (SQ) adipocytes derived from the same human subjects. To define global differences in gene expression patterns between PV and SQ adipocytes, genome-wide microarray studies were performed in three sets of in vitro differentiated SQ and PV adipocytes derived from unrelated human subjects. Insulin-regulated and Wnt signaling genes were markedly down-regulated in PV adipocytes. Validation of microarray data by qPCR demonstrated reductions in expression of C/EBPα, PPARγ, FABP4, adiponectin, lipoprotein lipase, hormone sensitive lipase and perilipin in PV compared to SQ adipocytes. We further observed that insulin-induced Akt ser-473 phosphorylation and glucose uptake were markedly reduced (∼ 3 fold and 4 fold, respectively) in differentiated PV adipocytes compared to SQ adipocytes. The mRNA levels of insulin and insulin-like growth factor receptors, however, were similar in adipocytes differentiated from these two depots. Regarding the Wnt pathway, PV adipocytes exhibited dramatically elevated expression of Wnt inhibitor DKK1 (2864%) and reduced expression of Wnt 5A (50%), FDZ4 (38%), and LRP5 (38%). Further evaluation revealed that these Wnt signaling pathway genes, like those of the insulin signaling pathway, correlated with the extent of adipogenic differentiation. We propose that dysregulation of Wnt 5A/FDZ4 and insulin signaling pathways contributes to impaired adipogenic differentiation and insulin resistance in PV adipocytes. This, in turn, may contribute to heightened inflammatory crosstalk between PV adipose tissue and the vascular wall in the setting of atherosclerosis.

The low-density lipoprotein receptor-related protein 8 (Lrp8; apolipoprotein E receptor 2; ApoER2) is a member of the low-density lipoprotein receptor ( Ldlr ) gene family which is predominately expressed in the brain, but is also expressed in vascular cells and macrophages where its function is currently unknown. Genomewide linkage analysis has associated a single nucleotide polymorphism in the LRP8 gene with familial and premature coronary artery disease and myocardial infarction in humans, suggesting that Lrp8 may contribute to atherosclerosis. This study assessed the role of Lrp8 in macrophage function and atherosclerosis. Following 24 weeks on a high-fat, high-cholesterol Western-type diet, lesion sizes were similar between Lrp8 knockout mice on an Ldlr deficient background ( Lrp8 -/- Ldlr -/- ) and Ldlr single knockout ( Ldlr -/- ) mice, but more complex lesions with extensive necrosis were observed in the Lrp8 -/- Ldlr -/- mice. The differences in lesion composition were not due to differences in plasma cholesterol levels, but increased expression of the macrophage marker CD68 in the Lrp8 -/- Ldlr -/- lesions suggested that these differences were due to alterations in macrophage function. When compared to wild-type (WT) cells, mRNA levels of the nuclear receptor PPARγ and PPARγ-responsive genes such as Cd36, Abca1 , and Lrp1 were significantly increased in Lrp8 deficient macrophages. Additionally, the expression of inducible nitric oxide synthase (iNOS) and Ldlr were reduced in Lrp8 deficient macrophages compared to WT cells. Furthermore, following 24 hours in culture there was a reduced number of adherent macrophages found in the Lrp8 deficient population compared to the WT cells. Consistent with this, phosphorylation of Akt was reduced while phosphorylation of p53 was increased in Lrp8 deficient macrophages, suggesting that Lrp8 may affect macrophage viability and proliferation via modulation of cell signaling events. Taken together, these data suggest that Lrp8 plays a critical role in macrophage function via regulation of foam cell formation as well as cell proliferation and viability, with reduced expression of Lrp8 significantly contributing to the pathogenesis of atherosclerosis and lesion necrosis.

Human high density lipoproteins (HDL) are divided into two major subpopulations based on lipoprotein composition. The predominant subpopulation contains both major apolipoproteins apoA-I and apoA-II (termed LpA-I/A-II), while the minor subpopulation contains only apoA-I (termed LpA-I). It is speculated that LpA-I/A-II could demonstrate different anti-oxidative and anti-inflammatory properties compared to LpA-I. The goal of the current work was to dissect out any contributions from apoA-II on such beneficial functions of HDL. We generated a series of reconstituted (r)LpA-I/A-II in which major apolipoprotein ratios mimic native LpA-I/A-II (apoA-I: apoA-II; 2:1, 2:2 and 2:3) but lack the anti-oxidative enzymes in native HDL. First, we generated the precursor rLpA-I by reacting discoidal HDL with recombinant lecithin cholesterol acyl transferase (LCAT) in the presence of low density lipoproteins. The rLpA-I consist of a single population of ∼93 Å particles. Cross-linking followed by MALDI-MS indicated exactly three apoA-I per rLpA-I. Different rLpA-I/A-II complexes were generated by adding the required amount of apoA-II into rLpA-I followed by isolation of rLpA-I:A-II using size exclusion chromatography. All rLpA-I/A-II showed comparable particle diameter to rLpA-I. Electron microscopy clearly captured the spherical nature of these particles and agarose gel electrophoresis demonstrated the presence of a neutral lipid core in rLpA-I and rLpA-I/A-II compared to discoidal HDL. All particles contain comparable amounts of total protein 36-40%, phospholipids 40-45%, cholesteryl esters 17-22%, free cholesterol

Objective— Adiponectin is an adipocyte-derived, secreted protein that is implicated in protection against a cluster of related metabolic disorders. Mice lacking adiponectin display impaired hepatic insulin sensitivity and respond only partially to peroxisome proliferator-activated receptor γ agonists. Adiponectin has been associated with antiinflammatory and antiatherogenic properties; however, the direct involvement of adiponectin on the atherogenic process has not been studied. Methods and Results— We crossed adiponectin knockout mice ( Adn −/− ) or mice with chronically elevated adiponectin levels ( Adn Tg ) into the low-density lipoprotein receptor–null ( Ldlr −/− ) and the apoliprotein E–null ( Apoe −/− ) mouse models. Adiponectin levels did not correlate with a suppression of the atherogenic process. Plaque volume in the aortic root, cholesterol accumulation in the aorta, and plaque morphology under various dietary conditions were not affected by circulating adiponectin levels. In light of the strong associations reported for adiponectin with cardiovascular disease in humans, the lack of a phenotype in gain- and loss-of-function studies in mice suggests a lack of causation for adiponectin in inhibiting the buildup of atherosclerotic lesions. Conclusion— These data indicate that the actions of adiponectin on the cardiovascular system are complex and multifaceted, with a minimal direct impact on atherosclerotic plaque formation in preclinical rodent models.

Five inbred strains of mice differing in susceptibility to diet-induced atherosclerosis were compared for neointimal hyperplasia after endothelial denudation with an epoxy resin–modified catheter probe. Results showed that all animals responded similarly to the arterial injury, with increased medial area and thickness after 14 days. In contrast, a significant strain-specific difference in neointimal formation after injury was observed. The atherosclerosis-susceptible C57L/J mice were also susceptible to injury-induced neointimal hyperplasia, and the C3H mice were resistant to both forms of vascular diseases. The 129/Sv mice, which displayed an intermediate level of diet-induced atherosclerosis, also displayed an intermediate level of injury-induced neointimal hyperplasia. Interestingly, the atherosclerosis-susceptible C57BL/6 mice were resistant to neointimal hyperplasia after endothelial denudation, whereas the atherosclerosis-resistant FVB/N mice were susceptible, displaying massive neointimal hyperplasia after arterial injury. All (C57L/J×C57BL/6)F 1 hybrid mice were resistant to injury-induced neointimal hyperplasia. Moreover, N 2 mice generated from backcrossing the F 1 hybrid mice to the susceptible C57L/J mice displayed a range of arterial response to injury, spanning the most severe to the most resistant phenotype. These results indicate that injury-induced neointimal hyperplasia and diet-induced atherosclerosis are controlled by distinct sets of genes; the former appeared to be determined by recessive genes at ≥2 loci.