Showing papers in "Arteriosclerosis, Thrombosis, and Vascular Biology in 2014"

Smoking and cardiovascular disease: mechanisms of endothelial dysfunction and early atherogenesis.

Abstract: Smoking represents one of the most important preventable risk factors for the development of atherosclerosis. The present review aims at providing a comprehensive summary of published data from clinical and animal studies, as well as results of basic research on the proatherogenic effect of smoking. Extensive search and review of literature revealed a vast amount of data on the influence of cigarette smoke and its constituents on early atherogenesis, particularly on endothelial cells. Vascular dysfunction induced by smoking is initiated by reduced nitric oxide (NO) bioavailability and further by the increased expression of adhesion molecules and subsequent endothelial dysfunction. Smoking-induced increased adherence of platelets and macrophages provokes the development of a procoagulant and inflammatory environment. After transendothelial migration and activation, macrophages take up oxidized lipoproteins arising from oxidative modifications and transdifferentiate into foam cells. In addition to direct physical damage to endothelial cells, smoking induces tissue remodeling, and prothrombotic processes together with activation of systemic inflammatory signals, all of which contribute to atherogenic vessel wall changes. There are still great gaps in our knowledge about the effects of smoking on cardiovascular disease. However, we know that smoking cessation is the most effective measure for reversing damage that has already occurred and preventing fatal cardiovascular outcomes.

Thrombosis A Major Contributor to Global Disease Burden

Abstract: Background— Thrombosis is the common pathology underlying ischemic heart disease, ischemic stroke, and venous thromboembolism (VTE). The Global Burden of Disease Study 2010 (GBD 2010) documented that ischemic heart disease and stroke collectively caused 1 in 4 deaths worldwide. GBD 2010 did not report data for VTE as a cause of death and disability. Objective— To review the literature on the global burden of disease caused by VTE. Approach and Results— We performed a systematic review of the literature on the global disease burden because of VTE in low-, middle-, and high-income countries. Studies from Western Europe, North America, Australia, and Southern Latin America (Argentina) yielded consistent results with annual incidences ranging from 0.75 to 2.69 per 1000 individuals in the population. The incidence increased to between 2 and 7 per 1000 among those aged ≥70 years. Although the incidence is lower in individuals of Chinese and Korean ethnicity, their disease burden is not low because of population aging. VTE associated with hospitalization was the leading cause of disability-adjusted life-years lost in low- and middle-income countries, and second in high-income countries, responsible for more disability-adjusted life-years lost than nosocomial pneumonia, catheter-related blood stream infections, and adverse drug events. Conclusions— VTE causes a major burden of disease across low-, middle-, and high-income countries. More detailed data on the global burden of VTE should be obtained to inform policy and resource allocation in health systems and to evaluate whether improved use of preventive measures will reduce the burden.

Oxidized low-density lipoprotein induces long-term proinflammatory cytokine production and foam cell formation via epigenetic reprogramming of monocytes

Abstract: Objective— Although the role of monocytes in the pathogenesis of atherosclerosis is well established, the persistent vascular inflammation remains largely unexplained. Recently, our group reported that stimulation of monocytes with various microbial products can induce a long-lasting proinflammatory phenotype via epigenetic reprogramming, a process termed trained immunity. We now hypothesize that oxidized low-density lipoprotein (oxLDL) also induces a long-lasting proinflammatory phenotype in monocytes, which accelerates atherosclerosis by proinflammatory cytokine production and foam cell formation. Approach and Results— Isolated human monocytes were exposed for 24 hours to medium or oxLDL. After washing and resting for 6 days, the cells were exposed to toll-like receptor 2 and 4 agonists. Pre-exposure to oxLDL increased mRNA expression and protein formation on toll-like receptor 2 and 4 stimulation of several proatherogenic proteins, including interleukin-6, interleukin-18, interleukin-8, tumor necrosis factor-α, monocyte chemoattractant protein 1, and matrix metalloproteinase 2 and 9. In addition, foam cell formation was enhanced after oxLDL exposure, which was associated with an upregulation of scavenger receptors CD36 and scavenger receptor-A and downregulation of ATP-binding cassette transporters, ABCA1 and ABCG1. Chromatin immunoprecipitation performed 6 days after oxLDL stimulation demonstrated increased trimethylation of lysine 4 at histone 3 in promoter regions of tnfα , il-6 , il-18 , mcp-1 , mmp2 , mmp9 , cd36 , and sr-a. Finally, pretreatment of the monocytes with the histone methyltransferase inhibitor methylthioadenosine completely prevented the oxLDL-induced long-lasting proinflammatory phenotype. Conclusions— Brief exposure of monocytes to a low concentration of oxLDL induces a long-lasting proatherogenic macrophage phenotype via epigenetic histone modifications, characterized by increased proinflammatory cytokine production and foam cell formation. # Significance {#article-title-30}

Neutrophil Extracellular Traps Promote Thrombin Generation Through Platelet-Dependent and Platelet-Independent Mechanisms

Abstract: Objective—Activation of neutrophils by microbial or inflammatory stimuli results in the release of neutrophil extracellular traps (NETs) that are composed of DNA, histones, and antimicrobial proteins. In purified systems, cell-free DNA (CFDNA) activates the intrinsic pathway of coagulation, whereas histones promote thrombin generation through platelet-dependent mechanisms. However, the overall procoagulant effects of CFDNA/histone complexes as part of intact NETs are unknown. In this study, we examined the procoagulant potential of intact NETs released from activated neutrophils. We also determined the relative contribution of CFDNA and histones to thrombin generation in plasmas from patients with sepsis. Approach and Results—NETs released from phorbyl myristate–activated neutrophils enhance thrombin generation in platelet-poor plasma. This effect was DNA dependent (confirmed by DNase treatment) and occurred via the intrinsic pathway of coagulation (confirmed with coagulation factor XII– and coagulation f...

Has Our Understanding of Calcification in Human Coronary Atherosclerosis Progressed

Abstract: Coronary artery calcification is a well-established predictor of future cardiac events; however, it is not a predictor of unstable plaque. The intimal calcification of the atherosclerotic plaques may begin with smooth muscle cell apoptosis and release of matrix vesicles and is almost always seen microscopically in pathological intimal thickening, which appears as microcalcification (≥0.5 μm, typically 2, <5 mm) calcification. The calcification in thin-cap fibroatheromas and plaque rupture is generally less than what is observed in stable plaques and is usually speckled or fragmented. Fragmented calcification spreads into the surrounding collagen-rich matrix forming calcified sheets, the hallmarks of fibrocalcific plaques. The calcified sheets may break into nodules with fibrin deposition, and when accompanied by luminal protrusion, it is associated with thrombosis. Calcification is highest in fibrocalcific plaques followed by healed plaque rupture and is the least in erosion and pathological intimal thickening. The extent of calcification is greater in men than in women especially in the premenopausal period and is also greater in whites compared with blacks. The mechanisms of intimal calcification remain poorly understood in humans. Calcification often occurs in the presence of apoptosis of smooth muscle cells and macrophages with matrix vesicles accompanied by expression of osteogenic markers within the vessel wall.

Small Dense Low-Density Lipoprotein-Cholesterol Concentrations Predict Risk for Coronary Heart Disease The Atherosclerosis Risk in Communities (ARIC) Study

Abstract: Objective—To investigate the relationship between plasma levels of small dense low-density lipoprotein-cholesterol (sdLDL-C) and risk for incident coronary heart disease (CHD) in a prospective study among Atherosclerosis Risk in Communities (ARIC) study participants. Approach and Results—Plasma sdLDL-C was measured in 11 419 men and women of the biracial ARIC study using a newly developed homogeneous assay. A proportional hazards model was used to examine the relationship among sdLDL-C, vascular risk factors, and risk for CHD events (n=1158) for a period of ≈11 years. Plasma sdLDL-C levels were strongly correlated with an atherogenic lipid profile and were higher in patients with diabetes mellitus than non–diabetes mellitus (49.6 versus 42.3 mg/dL; P<0.0001). In a model that included established risk factors, sdLDL-C was associated with incident CHD with a hazard ratio of 1.51 (95% confidence interval, 1.21–1.88) for the highest versus the lowest quartile, respectively. Even in individuals considered to b...

Shear Stress–Initiated Signaling and Its Regulation of Endothelial Function

Abstract: Atherosclerosis develops preferentially at branches and curvatures of the arterial tree, where blood flow pattern is disturbed rather than being laminar, and wall shear stress has an irregular distribution without defined directions. The endothelium in the atherosusceptible regions, in comparison to that in atheroresistant regions, shows activation of proproliferative and proinflammatory gene expressions, reduced production of nitric oxide (NO), increased leukocyte adhesion, and permeability, as well as other atheroprone phenotypes. Differences in gene expressions and cell phenotypes have been detected in endothelia residing in native atherosusceptible and atheroresistant regions of the arteries, or in arteries from animal models with artificial creation of disturbed flow. Similar results have also been shown in in vitro systems that apply controlled shear stresses with or without clear directions to cultured endothelial cells in fluid-dynamically designed flow-loading devices. The available evidence indicates that the coordination of multiple signaling networks, rather than individual separate pathways, links the mechanical signals to specific genetic circuitries in orchestrating the mechanoresponsive networks to evoke comprehensive genetic and functional responses.

Inflammatory, metabolic, and genetic mechanisms of vascular calcification.

Abstract: This review centers on updating the active research area of vascular calcification. This pathology underlies substantial cardiovascular morbidity and mortality, through adverse mechanical effects on vascular compliance, vasomotion, and, most likely, plaque stability. Biomineralization is a complex, regulated process occurring widely throughout nature. Decades ago, its presence in the vasculature was considered a mere curiosity and an unregulated, dystrophic process that does not involve biological mechanisms. Although it remains controversial whether the process has any adaptive value or past evolutionary advantage, substantial advances have been made in understanding the biological mechanisms driving the process. Different types of calcific vasculopathy, such as inflammatory versus metabolic, have parallel mechanisms in skeletal bone calcification, such as intramembranous and endochondral ossification. Recent work has identified important regulatory roles for inflammation, oxidized lipids, elastin, alkaline phosphatase, osteoprogenitor cells, matrix γ-carboxyglutamic acid protein, transglutaminase, osteoclastic regulatory factors, phosphate regulatory hormones and receptors, apoptosis, prelamin A, autophagy, and microvesicles or microparticles similar to the matrix vesicles of skeletal bone. Recent work has uncovered fascinating interactions between matrix γ-carboxyglutamic acid protein, vitamin K, warfarin, and transport proteins. And, lastly, recent breakthroughs in inherited forms of calcific vasculopathy have identified the genes responsible as well as an unexpected overlap of phenotypes. Until recently, vascular calcification was considered a purely degenerative, unregulated process. Since then, investigative groups around the world have identified a wide range of causative mechanisms and regulatory pathways, and some of the recent developments are highlighted in this review.

T-Cell Profile in Adipose Tissue Is Associated With Insulin Resistance and Systemic Inflammation in Humans

Abstract: Objective— The biological mechanisms linking obesity to insulin resistance have not been fully elucidated. We have shown that insulin resistance or glucose intolerance in diet-induced obese mice is related to a shift in the ratio of pro- and anti-inflammatory T cells in adipose tissue. We sought to test the hypothesis that the balance of T-cell phenotypes would be similarly related to insulin resistance in human obesity. Approach and Results— Healthy overweight or obese human subjects underwent adipose-tissue biopsies and quantification of insulin-mediated glucose disposal by the modified insulin suppression test. T-cell subsets were quantified by flow cytometry in visceral (VAT) and subcutaneous adipose tissue (SAT). Results showed that CD4 and CD8 T cells infiltrate both depots, with proinflammatory T-helper (Th)-1, Th17, and CD8 T cells, significantly more frequent in VAT as compared with SAT. T-cell profiles in SAT and VAT correlated significantly with one another and with peripheral blood. Th1 frequency in SAT and VAT correlated directly, whereas Th2 frequency in VAT correlated inversely, with plasma high-sensitivity C-reactive protein concentrations. Th2 in both depots and peripheral blood was inversely associated with systemic insulin resistance. Furthermore, Th1 in SAT correlated with plasma interleukin-6. Relative expression of associated cytokines, measured by real-time polymerase chain reaction, reflected flow cytometry results. Most notably, adipose tissue expression of anti-inflammatory interleukin-10 was inversely associated with insulin resistance. Conclusions— CD4 and CD8 T cells populate human adipose tissue and the relative frequency of Th1 and Th2 are highly associated with systemic inflammation and insulin resistance. These findings point to the adaptive immune system as a potential mediator between obesity and insulin resistance or inflammation. Identification of antigenic stimuli in adipose tissue may yield novel targets for treatment of obesity-associated metabolic disease. # Significance {#article-title-25}

Abstract 53: An Abundant Dysfunctional Apolipoprotein A1 in Human Atheroma

Abstract: High density lipoproteins (HDL) are a heterogeneous group of particles formed predominantly around the major structural protein, apolipoprotein A1 (apoA1). Contemporary findings suggest functional measures of HDL/apoA1 may provide improved clinical relevance. Moreover, recent studies indicate apoA1 in human atheroma is dysfunctional, extensively oxidized by myeloperoxidase (MPO)-generated oxidants, and that in vitro oxidation of apoA1 by MPO impairs its cholesterol acceptor function. Using phage-display affinity maturation and recombinant immunoglobulin engineering we developed a high affinity monoclonal antibody (mAb) that specifically recognizes apoA1/HDL modified by the MPO/H 2 O 2 /halide system. An oxindolyl alanine (2-OH-Trp) moiety at tryptophan 72 of apoA1 serves as the mAb recognition site. Mutagenesis studies confirm a critical role for apoA1 Trp72 in MPO-dependent inhibition of ABCA1-dependent cholesterol acceptor activity of apoA1 in vitro and in vivo. While apoA1 containing a 2-OH-Trp 72 group (oxTrp72apoA1) is in low abundance within the circulation, it is remarkably abundant within human atherosclerotic plaque, accounting for 20% of apoA1 in aortic lesions. Functional characterization of immune-purified oxTrp72apoA1 from human atherosclerotic plaque and plasma reveals a lipid-poor lipoprotein virtually devoid of cholesterol acceptor activity. Recovered oxTrp72apoA1 demonstrated impaired capacity to promote HDL biogenesis in vivo, and potent pro-inflammatory activities including NF-κB activation and VCAM-1 surface expression on endothelial cells. Elevated oxTrp72apoA1 levels among sequential subjects presenting to a preventive cardiology clinic (n=627) were associated with increased cardiovascular disease risk, including after adjustments for traditional risk factors, apoA1 and MPO [Odds ratio (95%confidence interval), 4.2(1.5-13.0)]. Circulating oxTrp72apoA1 levels provide a window into monitoring the generation of an abundant pro-atherogenic apoA1 form within the artery wall.

Calcific Aortic Valve Disease: A Consensus Summary From the Alliance of Investigators on Calcific Aortic Valve Disease

Abstract: Calcific aortic valve disease (CAVD) is increasingly prevalent worldwide with significant morbidity and mortality. Therapeutic options beyond surgical valve replacement are currently limited. In 2011, the National Heart Lung and Blood Institute assembled a working group on aortic stenosis. This group identified CAVD as an actively regulated disease process in need of further study. As a result, the Alliance of Investigators on CAVD was formed to coordinate and promote CAVD research, with the goals of identifying individuals at risk, developing new therapeutic approaches, and improving diagnostic methods. The group is composed of cardiologists, geneticists, imaging specialists, and basic science researchers. This report reviews the current status of CAVD research and treatment strategies with identification of areas in need of additional investigation for optimal management of this patient population.

Identification and Initial Functional Characterization of a Human Vascular Cell–Enriched Long Noncoding RNA

Abstract: Objective— Long noncoding RNAs (lncRNAs) represent a rapidly growing class of RNA genes with functions related primarily to transcriptional and post-transcriptional control of gene expression. There is a paucity of information about lncRNA expression and function in human vascular cells. Thus, we set out to identify novel lncRNA genes in human vascular smooth muscle cells and to gain insight into their role in the control of smooth muscle cell phenotypes. Approach and Results— RNA sequencing of human coronary artery smooth muscle cells revealed 31 unannotated lncRNAs, including a vascular cell–enriched lncRNA (smooth muscle and endothelial cell–enriched migration/differentiation-associated long noncoding RNA [ SENCR ]). Strand-specific reverse transcription polymerase chain reaction (PCR) and rapid amplification of cDNA ends indicate that SENCR is transcribed antisense from the 5′ end of the FLI1 gene and exists as 2 splice variants. RNA fluorescence in situ hybridization and biochemical fractionation studies demonstrate SENCR is a cytoplasmic lncRNA. Consistent with this observation, knockdown studies reveal little to no cis -acting effect of SENCR on FLI1 or neighboring gene expression. RNA-sequencing experiments in smooth muscle cells after SENCR knockdown disclose decreased expression of Myocardin and numerous smooth muscle contractile genes, whereas several promigratory genes are increased. Reverse transcription PCR and Western blotting experiments validate several differentially expressed genes after SENCR knockdown. Loss-of-function studies in scratch wound and Boyden chamber assays support SENCR as an inhibitor of smooth muscle cell migration. Conclusions— SENCR is a new vascular cell–enriched, cytoplasmic lncRNA that seems to stabilize the smooth muscle cell contractile phenotype.

Perivascular Adipose Tissue in Vascular Function and Disease: A Review of Current Research and Animal Models

Abstract: Perivascular adipose tissue (PVAT), long assumed to be nothing more than vessel-supporting connective tissue, is now understood to be an important, active component of the vasculature, with integral roles in vascular health and disease. PVAT is an adipose tissue with similarities to both brown and white adipose tissue, although recent evidence suggests that PVAT develops from its own precursors. Like other adipose tissue depots, PVAT secretes numerous biologically active substances that can act in both autocrine and paracrine fashion. PVAT has also proven to be involved in vascular inflammation. Although PVAT can support inflammation during atherosclerosis via macrophage accumulation, emerging evidence suggests that PVAT also has antiatherosclerotic properties related to its abilities to induce nonshivering thermogenesis and metabolize fatty acids. We here discuss the accumulated knowledge of PVAT biology and related research on models of hypertension and atherosclerosis.

ABCC6–Mediated ATP Secretion by the Liver Is the Main Source of the Mineralization Inhibitor Inorganic Pyrophosphate in the Systemic Circulation—Brief Report

Abstract: Objective—Mutations in ABCC6 underlie the ectopic mineralization disorder pseudoxanthoma elasticum (PXE) and some forms of generalized arterial calcification of infancy, both of which affect the cardiovascular system. Using cultured cells, we recently showed that ATP-binding cassette subfamily C member 6 (ABCC6) mediates the cellular release of ATP, which is extracellularly rapidly converted into AMP and the mineralization inhibitor inorganic pyrophosphate (PPi). The current study was performed to determine which tissues release ATP in an ABCC6-dependent manner in vivo, where released ATP is converted into AMP and PPi, and whether human PXE ptients have low plasma PPi concentrations. Approach and Results—Using cultured primary hepatocytes and in vivo liver perfusion experiments, we found that ABCC6 mediates the direct, sinusoidal, release of ATP from the liver. Outside hepatocytes, but still within the liver vasculature, released ATP is converted into AMP and PPi. The absence of functional ABCC6 in patien...

Role of Flow-Sensitive microRNAs in Endothelial Dysfunction and Atherosclerosis Mechanosensitive Athero-miRs

Abstract: Atherosclerosis preferentially occurs in arterial regions exposed to disturbed flow, in part, due to alterations in gene expression. MicroRNAs (miRNAs) are small, noncoding genes that post-transcriptionally regulate gene expression by targeting messenger RNA transcripts. Emerging evidence indicates that alteration of flow conditions regulate expression of miRNAs in endothelial cells both in vitro and in vivo. These flow-sensitive miRNAs, known as mechano-miRs, regulate endothelial gene expression and can regulate endothelial dysfunction and atherosclerosis. MiRNAs such as, miR-10a, miR-19a, miR-23b, miR-17-92, miR-21, miR-663, miR-92a, miR-143/145, miR-101, miR-126, miR-712, miR-205, and miR-155, have been identified as mechano-miRs. Many of these miRNAs were initially identified as flow sensitive in vitro and were later found to play a critical role in endothelial function and atherosclerosis in vivo through either gain-of-function or loss-of-function approaches. The key signaling pathways that are targeted by these mechano-miRs include the endothelial cell cycle, inflammation, apoptosis, and nitric oxide signaling. Furthermore, we have recently shown that the miR-712/205 family, which is upregulated by disturbed flow, contributes to endothelial inflammation and vascular hyperpermeability by targeting tissue inhibitor of metalloproteinase-3, which regulates metalloproteinases and a disintegrin and metalloproteinases. The mechano-miRs that are implicated in atherosclerosis are termed as mechanosensitive athero-miRs and are potential therapeutic targets to prevent or treat atherosclerosis. This review summarizes the current knowledge of mechanosensitive athero-miRs and their role in vascular biology and atherosclerosis.

Interleukin 17 Drives Vascular Inflammation, Endothelial Dysfunction, and Arterial Hypertension in Psoriasis-Like Skin Disease

Abstract: Objective— Interleukin (IL)-17A is regarded as an important cytokine to drive psoriasis, an inflammatory skin disease marked by increased cardiovascular mortality. We aimed to test the hypothesis that overproduction of IL-17A in the skin leading to dermal inflammation may systemically cause vascular dysfunction in psoriasis-like skin disease. Approach and Results— Conditional overexpression of IL-17A in keratinocytes caused severe psoriasis-like skin inflammation in mice (K14-IL-17Aind/+ mice), associated with increased reactive oxygen species formation and circulating CD11b+ inflammatory leukocytes in blood, with endothelial dysfunction, increased systolic blood pressure, left ventricular hypertrophy, and reduced survival compared with controls. In K14-IL-17Aind/+ mice, immunohistochemistry and flow cytometry revealed increased vascular production of the nitric oxide/superoxide reaction product peroxynitrite and infiltration of the vasculature with myeloperoxidase+CD11b+GR1+F4/80− cells accompanied by increased expression of the inducible nitric oxide synthase and the nicotinamide dinucleotide phosphate (NADPH) oxidase, nox2. Neutrophil depletion by anti-GR-1 antibody injections reduced oxidative stress in blood and vessels. Neutralization of tumor necrosis factor-α and IL-6 (both downstream of IL-17A) reduced skin lesions, attenuated oxidative stress in heart and blood, and partially improved endothelial dysfunction in K14-IL-17Aind/+ mice. Conclusions— Dermal overexpression of IL-17A induces systemic endothelial dysfunction, vascular oxidative stress, arterial hypertension, and increases mortality mainly driven by myeloperoxidase+CD11b+GR1+F4/80− inflammatory cells. Depletion of the GR-1+ immune cells or neutralization of IL-17A downstream cytokines by biologicals attenuates the vascular phenotype in K14-IL-17Aind/+ mice. # Significance {#article-title-54}

Disturbed Flow Promotes Endothelial Senescence via a p53-Dependent Pathway

Abstract: Objective— Although atherosclerosis is associated with systemic risk factors such as age, high cholesterol, and obesity, plaque formation occurs predominately at branches and bends that are exposed to disturbed patterns of blood flow. The molecular mechanisms that link disturbed flow–generated mechanical forces with arterial injury are uncertain. To illuminate them, we investigated the effects of flow on endothelial cell (EC) senescence. Approach and Results— LDLR−/− (low-density lipoprotein receptor−/−) mice were exposed to a high-fat diet for 2 to 12 weeks (or to a normal chow diet as a control) before the assessment of cellular senescence in aortic ECs. En face staining revealed that senescence-associated β-galactosidase activity and p53 expression were elevated in ECs at sites of disturbed flow in response to a high-fat diet. By contrast, ECs exposed to undisturbed flow did not express senescence-associated β-galactosidase or p53. Studies of aortae from healthy pigs (aged 6 months) also revealed enhanced senescence-associated β-galactosidase staining at sites of disturbed flow. These data suggest that senescent ECs accumulate at disturbed flow sites during atherogenesis. We used in vitro flow systems to examine whether a causal relationship exists between flow and EC senescence. Exposure of cultured ECs to flow (using either an orbital shaker or a syringe-pump flow bioreactor) revealed that disturbed flow promoted EC senescence compared with static conditions, whereas undisturbed flow reduced senescence. Gene silencing studies demonstrated that disturbed flow induced EC senescence via a p53-p21 signaling pathway. Disturbed flow–induced senescent ECs exhibited reduced migration compared with nonsenescent ECs in a scratch wound closure assay, and thus may be defective for arterial repair. However, pharmacological activation of sirtuin 1 (using resveratrol or SRT1720) protected ECs from disturbed flow–induced senescence. Conclusions— Disturbed flow promotes endothelial senescence via a p53-p21–dependent pathway which can be inhibited by activation of sirtuin 1. These observations support the principle that pharmacological activation of sirtuin 1 may promote cardiovascular health by suppressing EC senescence at atheroprone sites. # Significance {#article-title-67}

Endothelial PFKFB3 Plays a Critical Role in Angiogenesis

Abstract: Objective— Vascular cells, particularly endothelial cells, adopt aerobic glycolysis to generate energy to support cellular functions. The effect of endothelial glycolysis on angiogenesis remains unclear. 6-Phosphofructo-2-kinase/fructose-2, 6-bisphosphatase, isoform 3 (PFKFB3) is a critical enzyme for endothelial glycolysis. By blocking or deleting PFKFB3 in endothelial cells, we investigated the influence of endothelial glycolysis on angiogenesis both in vitro and in vivo. Approach and Results— Under hypoxic conditions or after treatment with angiogenic factors, endothelial PFKFB3 was upregulated both in vitro and in vivo. The knockdown or overexpression of PFKFB3 suppressed or accelerated endothelial proliferation and migration in vitro, respectively. Neonatal mice from a model of oxygen-induced retinopathy showed suppressed neovascular growth in the retina when endothelial PFKFB3 was genetically deleted or when the mice were treated with a PFKFB3 inhibitor. In addition, tumors implanted in mice deficient in endothelial PFKFB3 grew more slowly and were provided with less blood flow. A lower level of phosphorylated protein kinase B was observed in PFKFB3-knockdown endothelial cells, which was accompanied by a decrease in intracellular lactate. The addition of lactate to PFKFB3-knockdown cells rescued the suppression of endothelial proliferation and migration. Conclusions— The blockade or deletion of endothelial PFKFB3 decreases angiogenesis both in vitro and in vivo. Thus, PFKFB3 is a promising target for the reduction of endothelial glycolysis and its related pathological angiogenesis. # Significance {#article-title-38}

Induction of Lysosomal Biogenesis in Atherosclerotic Macrophages Can Rescue Lipid-Induced Lysosomal Dysfunction and Downstream Sequelae

Abstract: Objective—Recent reports of a proatherogenic phenotype in mice with macrophage-specific autophagy deficiency have renewed interest in the role of the autophagy-lysosomal system in atherosclerosis. Lysosomes have the unique ability to process both exogenous material, including lipids and autophagy-derived cargo such as dysfunctional proteins/organelles. We aimed to understand the effects of an atherogenic lipid environment on macrophage lysosomes and to evaluate novel ways to modulate this system. Approach and Results—Using a variety of complementary techniques, we show that oxidized low-density lipoproteins and cholesterol crystals, commonly encountered lipid species in atherosclerosis, lead to profound lysosomal dysfunction in cultured macrophages. Disruptions in lysosomal pH, proteolytic capacity, membrane integrity, and morphology are readily seen. Using flow cytometry, we find that macrophages isolated from atherosclerotic plaques also display features of lysosome dysfunction. We then investigated whe...

Functionality of endothelial cells and pericytes from human pluripotent stem cells demonstrated in cultured vascular plexus and zebrafish xenografts.

Abstract: Objective— Endothelial cells (ECs), pericytes, and vascular smooth muscle cells (vSMCs) are essential for vascular development, and their dysfunction causes multiple cardiovascular diseases. Primary vascular cells for research are, however, difficult to obtain. Human-induced pluripotent stem cells (hiPSCs) derived from somatic tissue are a renewable source of ECs and vSMCs; however, their use as disease models has been limited by low and inconsistent efficiencies of differentiation and the lack of phenotypic bioassays. Approach and Results— Here, we developed defined conditions for simultaneous derivation of ECs and pericytes with high efficiency from hiPSCs of different tissue origin. The protocol was equally efficient for all lines and human embryonic stem cells (hESCs). The ECs could undergo sequential passage and were phenotypically indistinguishable, exhibiting features of arterial-like embryonic ECs. Moreover, hiPSC-derived ECs formed an authentic vascular plexus when cocultured with hiPSC-derived pericytes. The coculture system recapitulated (1) major steps of vascular development including EC proliferation and primary plexus remodeling, and (2) EC-mediated maturation and acquisition of contractile vSMC phenotype by pericytes. In addition, hiPSC-derived ECs integrated into developing vasculature as xenografts in zebrafish. This contrasts with more widely used ECs from human umbilical vein, which form only unstable vasculature and were completely unable to integrate into zebrafish blood vessels. Conclusions— We demonstrate that vascular derivatives of hiPSC, such as ECs and pericytes, are fully functional and can be used to study defective endothelia–pericyte interactions in vitro for disease modeling and studies on tumor angiogenesis.

MicroRNA-155 Deficiency Results in Decreased Macrophage Inflammation and Attenuated Atherogenesis in Apolipoprotein E–Deficient Mice

Abstract: Objective— microRNA-155 (miR155) plays a critical role in immunity and macrophage inflammation. We aim to investigate the role of miR155 in atherogenesis. Approach and Results— Quantitative real-time polymerase chain reaction showed that miR155 was expressed in mouse and human atherosclerotic lesions. miR155 expression in macrophages was correlated positively with proinflammatory cytokine expression. Lentivirus-mediated overexpression of miR155 in macrophages enhanced their inflammatory response to lipopolysaccharide through targeting suppressor of cytokine signaling-1 and impaired cholesterol efflux from acetylated low-density lipoprotein–loaded macrophages, whereas deficiency of miR155 blunted macrophage inflammatory responses and enhanced cholesterol efflux possibly via enhancing lipid loading–induced macrophage autophagy. We next examined the atherogenesis in apolipoprotein E–deficient (apoE−/−) and miR155−/−/apoE−/− (double knockout) mice fed a Western diet. Compared with apoE−/− mice, the double knockout mice developed less atherosclerosis lesion in aortic root, with reduced neutral lipid content and macrophages. Flow cytometric analysis showed that there were increased number of regulatory T cells and reduced numbers of Th17 cells and CD11b+/Ly6Chigh cells in the spleen of double knockout mice. Peritoneal macrophages from the double knockout mice had significantly reduced proinflammatory cytokine expression and secretion both in the absence and presence of lipopolysaccharide stimulation. To determine whether miR155 in leukocytes contributes to atherosclerosis, we performed a bone marrow transplantation study. Deficiency of miR155 in bone marrow–derived cells suppressed atherogenesis in apoE−/− mice, demonstrating that hematopoietic cell–derived miR155 plays a critical role. Conclusions— miR155 deficiency attenuates atherogenesis in apoE−/− mice by reducing inflammatory responses of macrophages, enhancing macrophage cholesterol efflux and resulting in an antiatherogenic leukocyte profile. Targeting miR155 may be a promising strategy to halt atherogenesis. # Significance {#article-title-51}

Metformin Protects Endothelial Function in Diet-Induced Obese Mice by Inhibition of Endoplasmic Reticulum Stress Through 5′ Adenosine Monophosphate–Activated Protein Kinase–Peroxisome Proliferator–Activated Receptor δ Pathway

Abstract: Objective—5′ Adenosine monophosphate–activated protein kinase (AMPK) interacts with peroxisome proliferator–activated receptor δ (PPARδ) to induce gene expression synergistically, whereas the activation of AMPK inhibits endoplasmic reticulum (ER) stress. Whether the vascular benefits of antidiabetic drug metformin (AMPK activator) in diabetes mellitus and obesity is mediated by PPARδ remains unknown. We aim to investigate whether PPARδ is crucial for metformin in ameliorating ER stress and endothelial dysfunction induced by high-fat diet. Approach and Results—Acetylcholine-induced endothelium-dependent relaxation in aortae was measured on wire myograph. ER stress markers were determined by Western blotting. Superoxide production in mouse aortae and NO generation in mouse aortic endothelial cells were assessed by fluorescence imaging. Endothelium-dependent relaxation was impaired and ER stress markers and superoxide level were elevated in aortae from high-fat diet–induced obese mice compared with lean mice...

Nε-(Carboxymethyl)lysine-Receptor for Advanced Glycation End Product Axis Is a Key Modulator of Obesity-Induced Dysregulation of Adipokine Expression and Insulin Resistance

Abstract: Objective—Dysregulation of inflammatory adipokines by the adipose tissue plays an important role in obesity-associated insulin resistance. Pathways leading to this dysregulation remain largely unknown. We hypothesized that the receptor for advanced glycation end products (RAGE) and the ligand Ne-(carboxymethyl)lysine (CML) are increased in adipose tissue and, moreover, that activation of the CML–RAGE axis plays an important role in obesity-associated inflammation and insulin resistance. Approach and Results—In this study, we observed a strong CML accumulation and increased expression of RAGE in adipose tissue in obesity. We confirmed in cultured human preadipocytes that adipogenesis is associated with increased levels of CML and RAGE. Moreover, CML induced a dysregulation of inflammatory adipokines in adipocytes via a RAGE-dependent pathway. To test the role of RAGE in obesity-associated inflammation further, we constructed an obese mouse model that is deficient for RAGE (ie, RAGE–/–/LeptrDb–/– mice). RAG...

Chemokines in Atherosclerosis Proceedings Resumed

Abstract: Chemokines play important roles in atherosclerotic vascular disease. Expressed by not only cells of the vessel wall but also emigrated leukocytes, chemokines were initially discovered to direct leukocytes to sites of inflammation. However, chemokines can also exert multiple functions beyond cell recruitment. Here, we discuss novel and recently emerging aspects of chemokines and their involvement in atherosclerosis. While reviewing newly identified roles of chemokines and their receptors in monocyte and neutrophil recruitment during atherogenesis and atheroregression, we also revisit homeostatic functions of chemokines, including their roles in cell homeostasis and foam cell formation. The functional diversity of chemokines in atherosclerosis warrants a clear-cut mechanistic dissection and stage-specific assessment to better appreciate the full scope of their actions in vascular inflammation and to identify pathways that harbor the potential for a therapeutic targeting of chemokines in atherosclerosis.

Links Between Ectopic Fat and Vascular Disease in Humans

Abstract: The average of overweight individual can have differential fat depots in target organs or specific compartments of the body. This ectopic fat distribution may be more of a predictive factor for cardiovascular risk than obesity. Abdominal visceral obesity, a representative ectopic fat, is robustly associated with insulin resistance and cardiovascular risk. Fat depots in the liver and muscle tissue cause adverse cardiometabolic risk by affecting glucose and lipid metabolism. Pericardial fat and perivascular fat affect coronary atherosclerosis, cardiac function, and hemodynamics. Fat around the neck is associated with systemic vascular resistance. Fat around the kidney may increase blood pressure and induce albuminuria. Fat accumulation in or around the pancreas alters glucose metabolism, conferring cardiovascular risk. Ectopic fat may act as an active endocrine and paracrine organ that releases various bioactive mediators that influence insulin resistance, glucose and lipid metabolism, coagulation, and inflammation, which all contribute to cardiovascular risk. Because both obese and apparently lean individuals can have ectopic fat, regional fat distribution may play an important role in the development of cardiovascular diseases in both nonobese and obese people.

Proinflammatory Phenotype of Perivascular Adipocytes

Abstract: Perivascular adipose tissue (PVAT) directly abuts the lamina adventitia of conduit arteries and actively communicates with the vessel wall to regulate vascular function and inflammation. Mounting evidence suggests that the biological activities of PVAT are governed by perivascular adipocytes, a unique class of adipocyte with distinct molecular and phenotypic characteristics. Perivascular adipocytes surrounding human coronary arteries (pericoronary perivascular adipocytes) exhibit a reduced state of adipogenic differentiation and a heightened proinflammatory state, secreting ≤50-fold higher levels of the proinflammatory cytokine monocyte chemoattractant peptide-1 compared with adipocytes from other regional depots. Thus, perivascular adipocytes may contribute to upregulated inflammation of PVAT observed in atherosclerotic human blood vessels. However, perivascular adipocytes also secrete anti-inflammatory molecules such as adiponectin, and elimination of PVAT in rodent models has been shown to augment vascular disease, suggesting that some amount of PVAT is required to maintain vascular homeostasis. Evidence in animal models and humans suggests that inflammation of PVAT may be modulated by environmental factors, such as high-fat diet and tobacco smoke, which are relevant to atherosclerosis. These findings suggest that the inflammatory phenotype of PVAT is diverse depending on species, anatomic location, and environmental factors and that these differences are fundamentally important in determining a pathogenic versus protective role of PVAT in vascular disease. Additional research into the mechanisms that regulate the inflammatory balance of perivascular adipocytes may yield new insight into, and treatment strategies for, cardiovascular disease.

Platelets Mediate Oxidized Low-Density Lipoprotein–Induced Monocyte Extravasation and Foam Cell Formation

Abstract: Objective—A growing body of evidence indicates that platelets contribute to the onset and progression of atherosclerosis by modulating immune responses. We aimed to elucidate the effects of oxidized low-density lipoprotein (OxLDL) on platelet–monocyte interactions and the consequences of these interactions on platelet phagocytosis, chemokine release, monocyte extravasation, and foam cell formation. Approach and Results—Confocal microscopy and flow cytometric analysis revealed that in vitro and in vivo stimulation with OxLDL resulted in rapid formation of platelet–monocyte aggregates, with a preference for CD16+ monocyte subsets. This platelet–monocyte interaction facilitated OxLDL uptake by monocytes, in a process that involved platelet CD36–OxLDL interaction, release of chemokines, such as CXC motif ligand 4, direct platelet–monocyte interaction, and phagocytosis of platelets. Inhibition of cyclooxygenase with acetylsalicylic acid and antagonists of ADP receptors, P2Y1 and P2Y12, partly abrogated OxLDL-i...

Interleukin 1 Receptor 1 and Interleukin 1β Regulate Megakaryocyte Maturation, Platelet Activation, and Transcript Profile During Inflammation in Mice and Humans

Abstract: Objective— Interleukin 1 Receptor 1 (IL1R1) and its ligand, IL1β, are upregulated in cardiovascular disease, obesity, and infection. Previously, we reported a higher level of IL1R1 transcripts in platelets from obese individuals of the Framingham Heart Study (FHS) but its functional effect in platelets has never been described. Additionally, IL1β levels are increased in atherosclerotic plaques and in bacterial infections. The aim of this work is to determine whether IL1β, through IL1R1, can activate platelets and megakaryocytes to promote atherothrombosis. Approach and Results— We found that IL1β-related genes from platelets, as measured in 1819 FHS participants, were associated with increased body mass index, and a direct relationship was shown in wild-type mice fed a high-fat diet. Mechanistically, IL1β activated NFκB and MAPK signaling pathways in megakaryocytes. IL1β, through IL1R1, increased ploidy of megakaryocytes to 64 N or more by 2-fold over control. IL1β increased agonist-induced platelet aggregation by 1.2-fold with thrombin and 4.2-fold with collagen. IL1β increased adhesion to both collagen and fibrinogen, and heterotypic aggregation by 1.9-fold over resting. High-fat diet–enhanced platelet adhesion was absent in IL1R1 −/− mice. Wild-type mice infected with Porphyromonas gingivalis had circulating heterotypic aggregates (1.5-fold more than control at 24 hours and 6.2-fold more at 6 weeks) that were absent in infected IL1R1 −/− and IL1β −/− mice. Conclusions— In summary, IL1R1- and IL1β-related transcripts are elevated in the setting of obesity. IL1R1/IL1β augment both megakaryocyte and platelet functions, thereby promoting a prothrombotic environment during infection and obesity; potentially contributing to the development of atherothrombotic disease.

A Brief Etymology of the Collateral Circulation

Abstract: It is well known that the protective capacity of the collateral circulation falls short in many individuals with ischemic disease of the heart, brain, and lower extremities. In the past 15 years, opportunities created by molecular and genetic tools, together with disappointing outcomes in many angiogenic trials, have led to a significant increase in the number of studies that focus on: understanding the basic biology of the collateral circulation; identifying the mechanisms that limit the collateral circulation's capacity in many individuals; devising methods to measure collateral extent, which has been found to vary widely among individuals; and developing treatments to increase collateral blood flow in obstructive disease. Unfortunately, accompanying this increase in reports has been a proliferation of vague terms used to describe the disposition and behavior of this unique circulation, as well as the increasing misuse of well-ensconced ones by new (and old) students of collateral circulation. With this in mind, we provide a brief glossary of readily understandable terms to denote the formation, adaptive growth, and maladaptive rarefaction of collateral circulation. We also propose terminology for several newly discovered processes that occur in the collateral circulation. Finally, we include terms used to describe vessels that are sometimes confused with collaterals, as well as terms describing processes active in the general arterial-venous circulation when ischemic conditions engage the collateral circulation. We hope this brief review will help unify the terminology used in collateral research.

Ectopic Fat, Insulin Resistance, and Nonalcoholic Fatty Liver Disease: Implications for Cardiovascular Disease

Abstract: Ectopic fat accumulation in the liver causes nonalcoholic fatty liver disease (NAFLD), which is the most common cause of chronic liver disease in Western countries. Ectopic liver lipid, particularly diacylglycerol, exacerbates hepatic insulin resistance, promotes systemic inflammation, and increases risk of developing both type 2 diabetes mellitus and cardiovascular disease. Increasing evidence suggests that NAFLD is an emerging risk factor for cardiovascular disease, and although there are currently no licensed treatments for NAFLD per se, current evidence suggests that statin treatment is safe in NAFLD. Presently, there is insufficient evidence to indicate that statins or other cardioprotective agents, such as angiotensin receptor blockers, are effective in treating NAFLD. In this brief narrative review, we discuss the diagnosis of NAFLD and the role of ectopic liver fat to cause insulin resistance and to increase risk of both type 2 diabetes mellitus and cardiovascular disease. For this review, PubMed was searched for articles using the key words non-alcoholic fatty liver disease or fatty liver combined with diabetes risk, cardiovascular risk, and cardiovascular mortality between 1990 and 2014. Articles published in languages other than English were excluded.