Journals RSS : Gourt

Tyrosine Sulfation of Leukocyte Adhesion Molecules and Chemokine Receptors Promotes Atherosclerosis [Editorials]

Dimorphisms in the Membrane-Spanning Domain of EPCR Impact Systemic Coagulation [Editorials]

The Discovery of Cellular Immunity in the Atherosclerotic Plaque [History of Discovery]

It is now generally accepted that atherosclerosis is an inflammatory/immune disease triggered by LDL accumulation in the artery wall. When discovering T cells and the molecular components of a cellular immune response, we proposed that atherosclerosis is an inflammatory process with an autoimmune component. This notion was met with general skepticism but has gained support from experimental and clinical studies. Here we describe some of the early studies that helped developing this concept.

The Molecular Mechanisms of HDL and Associated Vesicular Trafficking Mechanisms to Mediate Cellular Lipid Homeostasis [History of Discovery]

HDL functions mainly as a cholesterol scavenger, facilitating transport of cholesterol to the liver for conversion to bile acids and secretion into the bile for elimination or recycling in the enterohepatic bile acid cycle. Because of its major function in cholesterol clearance, HDL is in general considered to be atheroprotective. From cell cholesterol can be removed by efflux especially to apoA-I and HDL as extracellular acceptors which transport the cholesterol to the liver for excretion. This process is called reverse cholesterol transport. In this context the ATP binding cassette transporter protein ABCA1 facilitates cellular cholesterol and phospholipid release to apoA-I-containing HDL precursors. In addition ABCA1 plays a role in vesicular lipid transport mechanisms required for HDL particle formation. In general to maintain intracellular lipid homeostasis, sterols and associated lipids move between cellular compartments by vesicular and nonvesicular pathways. However, cholesterol sorting on vesicle formation is poorly understood. This review summarizes the current knowledge of the molecular mechanisms of HDL and associated vesicular trafficking mechanisms to mediate cellular lipid homeostasis.

Adipose Tissue-Derived Stem Cells: Characterization and Potential for Cardiovascular Repair [Brief Reviews]

Experimental studies have shown that cardiac transfer of unfractionated or partially purified bone marrow cells, as well as stem cells and progenitor cells derived from the bone marrow or peripheral blood, can enhance functional recovery after an acute myocardial infarction. However, the relatively low abundance, small tissue volume, difficult accessibility, and disease-related malfunction of bone marrow-derived stem cells hamper their clinical usefulness. Numerous studies have provided evidence that stromal cells derived from the adipose tissue (adipose tissue-derived stromal cells [ADSCs]) contain a population of adult multipotent mesenchymal stem cells and endothelial progenitor cells that can differentiate into several lineages, including endothelial cells, smooth muscle cells, and cardiomyocytes. The similarities between stem cells extracted from the bone marrow and the adipose tissue suggest the potential for the adipose tissue to act as an alternative, and perhaps preferable, cell source for repairing damaged tissues, such as the ischemic or infarcted heart. We have here reviewed the medical literature describing molecular and functional characterization, differentiation, potential role, and results obtained so far using ADSCs in tissue repair, with a particular focus on the role for ADSCs in cardiovascular repair and regeneration.

Lack of Tyrosylprotein Sulfotransferase Activity in Hematopoietic Cells Drastically Attenuates Atherosclerosis in Ldlr-/- Mice [Integrative Physiology/Experimental Medicine]

Objective— Leukocyte recruitment is a major contributor in the development of atherosclerosis and requires a variety of proteins such as adhesion molecules, chemokines, and chemokine receptors. Several key molecular players implicated in this process are expressed on monocytes and require protein-tyrosine sulfation for optimal function in vitro, including human CCR2, CCR5, CX3CR1, and PSGL-1. We therefore hypothesized that protein-tyrosine sulfation in hematopoietic cells plays an important role in the development of atherosclerosis. Methods and Results— Lethally-irradiated Ldlr–/– mice were rescued with hematopoietic progenitors lacking tyrosylprotein sulfotransferase (TPST) activity attributable to deletion of the Tpst1 and Tpst2 genes. TPST deficient progenitors efficiently reconstituted hematopoiesis in Ldlr–/– recipients and transplantation had no effect on plasma lipids on a standard or atherogenic diet. However, we observed a substantial reduction in the size of atherosclerotic lesions and the number of macrophages in lesions from hyperlipidemic Ldlr–/– recipients transplanted with TPST deficient progenitors compared to wild-type progenitors. We also document for the first time that murine Psgl-1 and Cx3cr1 are tyrosine-sulfated. Conclusions— These data demonstrate that protein-tyrosine sulfation is an important contributor to monocytes/macrophage recruitment and/or retention in a mouse model of atherosclerosis.

Level of Macrophage uPA Expression Is an Important Determinant of Atherosclerotic Lesion Growth in Apoe-/- Mice [Integrative Physiology/Experimental Medicine]

Objective— Enhanced plasminogen activation, mediated by overexpression of urokinase-type plasminogen activator (uPA), accelerates atherosclerosis in apolipoprotein E–null mice. However, the mechanisms through which uPA acts remain unclear. In addition, although elevated uPA expression can accelerate murine atherosclerosis, there is not yet any evidence that decreased uPA expression would retard atherosclerosis. Methods and Results— We used a bone marrow transplant (BMT) approach and apolipoprotein E–deficient (Apoe–/–) mice to investigate cellular mechanisms of uPA-accelerated atherosclerosis, aortic dilation, and sudden death. We also used BMT to determine whether postnatal loss of uPA expression in macrophages retards atherosclerosis. BMT from uPA-overexpressing mice yielded recipients with macrophage-specific uPA overexpression; whereas BMT from uPA knockout mice yielded recipients with macrophage-specific loss of uPA expression. Recipients of uPA-overexpressing BM acquired all the vascular phenotypes (accelerated atherosclerosis, aortic medial destruction and dilation, severe coronary stenoses) as well as the sudden death phenotype of uPA-overexpressing mice. Moreover, fat-fed 37-week-old recipients of uPA-null BM had significantly less atherosclerosis than recipients of uPA wild-type marrow (40% less aortic surface lesion area; P=0.03). Conclusions— The level of uPA expression by macrophages—over a broad range—is an important determinant of atherosclerotic lesion growth in Apoe–/– mice.

Moderately Decreased Cholesterol Absorption Rates Are Associated With a Large Atheroprotective Effect [Integrative Physiology/Experimental Medicine]

Objective— Human treatment with ezetimibe results in a moderate 50% to 54% decrease in cholesterol absorption and a 15% to 20% decrease in plasma LDL-cholesterol levels; nevertheless, the efficacy of ezetimibe therapy has been recently challenged by the ENHANCE trial. We examined the efficacy of a moderate decrease in cholesterol absorption in preventing atherosclerosis formation in the mouse. Methods and Results— Congenic 14DKK animals, consisting of a castaneus (CASA/Rk) chromosome 14 interval introgressed onto the C57BL/6J background, displayed a moderate decrease in cholesterol absorption rates. The effect of moderately decreased absorption on atherosclerosis formation was determined in 14DKK apolipoprotein E knockouts (14DKK-apoEKO). When compared to chow diet–fed control apoEKO mice, congenic 14DKK-apoEKO displayed a moderate 41% decrease in cholesterol absorption rates, 30% to 37% decrease in plasma cholesterol levels, and a 70% decrease in atherosclerosis formation. Studies on cholesterol efflux and reverse cholesterol transport (RCT) from 14DKK bone marrow–derived macrophages rejected a 14DKK interval-dependent atheroprotective effects that operate in macrophages. In contrast, 14DKK-apoEKO congenics were characterized by a 60% increase in RCT from peripheral tissue macrophages. Conclusions— These studies strongly suggest that moderately decreased cholesterol absorption rates result in a large atheroprotective effect attributable to a decrease in plasma cholesterol levels and an increase in RCT from peripheral tissue macrophages.

Heat Shock Protein 27 Protects Against Atherogenesis via an Estrogen-Dependent Mechanism: Role of Selective Estrogen Receptor Beta Modulation [Integrative Physiology/Experimental Medicine]

Objective— We recently identified HSP27 as an atheroprotective protein that acts extracellularly to prevent foam cell formation and atherogenesis in female but not male mice, where serum levels of HSP27 were increased and inversely correlated with degree of lesion burden. In the current study we sought to determine whether estrogens are required for the observed atheroprotective benefits of HSP27 as well as its extracellular release. Methods and Results— In vitro estrogens prompted the release of HSP27 from macrophages in an ERβ specific manner that involved exosomal trafficking. Ovariectomy nullified the previously recognized attenuation in aortic lesion area in HSP27o/eapoE–/– mice compared to apoE–/– mice. Supplementation with 17β-estradiol resulted in a >15x increase in uterine weight and attenuation of atherogenesis in all mice, although HSP27o/eapoE–/– had 34% less lesion burden compared to apoE–/– mice. Mice treated with the ERβ-specific agonist, DPN had no effect on uterine weight but a 28% decrease in aortic lesion area in HSP27o/eapoE–/– compared to apoE–/– mice. HSP27 serum levels showed a similar gradual increase with E2 and DPN replacement treatment but did not change in untreated mice. Conclusions— The extracellular release of and atheroprotection provided by HSP27 is estrogen dependent.

Laminar Shear Stress Regulates Endothelial Kinin B1 Receptor Expression and Function: Potential Implication in Atherogenesis [Integrative Physiology/Experimental Medicine]

Objective— The proinflammatory phenotype induced by low laminar shear stress (LSS) is implicated in atherogenesis. The kinin B1 receptor (B1R), known to be induced by inflammatory stimuli, exerts many proinflammatory effects including vasodilatation and leukocyte recruitment. We investigated whether low LSS is a stimulus for endothelial B1R expression and function. Methods and Results— Human and mouse atherosclerotic plaques expressed high level of B1R mRNA and protein. In addition, B1R expression was upregulated in the aortic arch (low LSS region) of ApoE–/– mice fed a high-fat diet compared to vascular regions of high LSS and animals fed normal chow. Of interest, a greater expression of B1R was noticed in endothelial cells from regions of low LSS in aortic arch of ApoE–/– mice. B1R was also upregulated in human umbilical vein endothelial cells (HUVECs) exposed to low LSS (0 to 2 dyn/cm2) compared to physiological LSS (6 to 10 dyn/cm2): an effect similarly evident in murine vascular tissue perfused ex vivo. Functionally, B1R activation increased prostaglandin and CXCL5 expression in cells exposed to low, but not physiological, LSS. IL-1β and ox-LDL induced B1R expression and function in HUVECs, a response substantially enhanced under low LSS conditions and inhibited by blockade of NFB activation. Conclusion— Herein, we show that LSS is a major determinant of functional B1R expression in endothelium. Furthermore, whereas physiological high LSS is a powerful repressor of this inflammatory receptor, low LSS at sites of atheroma is associated with substantial upregulation, identifying this receptor as a potential therapeutic target.

Simvastatin Inhibits Angiotensin II-Induced Abdominal Aortic Aneurysm Formation in Apolipoprotein E-Knockout Mice: Possible Role of ERK [Integrative Physiology/Experimental Medicine]

Objective— Abdominal aortic aneurysm (AAA) is a life-threatening disease affecting almost 10% of the population over age 65. Generation of AAAs by infusion of angiotensin (Ang) II in apolipoprotein E-knockout (ApoE–/–) mice is an animal model which supports an imbalance of the renin-angiotensin system in the pathogenesis of AAA. The effect of statins on AngII-mediated AAA formation and the associated neovascularization is not known. Here we determined the effect of simvastatin and the ERK inhibitor, CI1040, on AngII-stimulated AAA formation. Methods and Results— ApoE–/– mice infused for 28 days with AngII using osmotic minipumps were treated with placebo, 10 mg/kg/d simvastatin, or 100 mg/kg/d CI1040. 95% of AngII-treated mice developed AAA with neovascularization of the lesion, increased ERK phosphorylation, MCP-1 secretion, and MMP activity. These effects were markedly reversed by simvastatin and in part by CI1040. Furthermore, simvastatin and the ERK inhibitor U0126 reversed AngII-stimulated angiogenesis and MMP secretion by human umbilical vein endothelial cells. Conclusions— These data support the conclusion that simvastatin interferes with AAA formation induced by AngII in ApoE–/– mice at least in part via ERK inhibition.

Smooth Muscle LDL Receptor-Related Protein-1 Inactivation Reduces Vascular Reactivity and Promotes Injury-Induced Neointima Formation [Integrative Physiology/Experimental Medicine]

Objective— Defective smooth muscle expression of LDL receptor-related protein-1 (Lrp1) increases atherosclerosis in hypercholesterolemic mice. This study explored the importance of smooth muscle Lrp1 expression under normolipidemic conditions. Methods and Results— Smooth muscle cells isolated from control (smLrp1+/+) and smooth muscle-specific Lrp1 knockout (smLrp1–/–) mice were characterized based on morphology, smooth muscle marker protein expression levels, and growth rates in vitro. Vascular functions were assessed by aortic constrictive response to agonist stimulation in situ and neointimal hyperplasia to carotid arterial injury in vivo. The smLrp1–/– smooth muscle cells displayed reduced -actin and calponin expression and an accelerated growth rate attribtuable to sustained phosphorylation of platelet-derived growth factor receptor (PRGFR) and protein kinase B/Akt. Vasoconstrictive response to agonist stimulation was impaired in aortic rings isolated from smLrp1–/– mice. Injury-induced neointimal hyperplasia was significantly increased in smLrp1–/– mice. The increase in neointima was associated with corresponding elevated activation of PDGFR signaling pathway. Conclusions— Smooth muscle expression of Lrp1 is important in maintaining normal vascular functions under normolipidemic conditions. The absence of Lrp1 expression results in greater smooth muscle cell proliferation, deficient contractile protein expression, impairment of vascular contractility, and promotion of denudation-induced neointimal hyperplasia.

Thiol Oxidative Stress Induced by Metabolic Disorders Amplifies Macrophage Chemotactic Responses and Accelerates Atherogenesis and Kidney Injury in LDL Receptor-Deficient Mice [Integrative Physiology/Experimental Medicine]

Background— Strengthening the macrophage glutathione redox buffer reduces macrophage content and decreases the severity of atherosclerotic lesions in LDL receptor-deficient (LDLR–/–) mice, but the underlying mechanisms were not clear. This study examined the effect of metabolic stress on the thiol redox state, chemotactic activity in vivo, and the recruitment of macrophages into atherosclerotic lesions and kidneys of LDL-R–/– mice in response to mild, moderate, and severe metabolic stress. Methods and Results— Reduced glutathione (GSH) and glutathione disulfide (GSSG) levels in peritoneal macrophages isolated from mildly, moderately, and severe metabolically-stressed LDL-R–/– mice were measured by HPLC, and the glutathione reduction potential (Eh) was calculated. Macrophage Eh correlated with the macrophage content in both atherosclerotic (r2=0.346, P=0.004) and renal lesions (r2=0.480, P=0.001) in these mice as well as the extent of both atherosclerosis (r2=0.414, P=0.001) and kidney injury (r2=0.480, P=0.001). Compared to LDL-R–/– mice exposed to mild metabolic stress, macrophage recruitment into MCP-1-loaded Matrigel plugs injected into LDL-R–/– mice increased 2.6-fold in moderately metabolically-stressed mice and 9.8-fold in severely metabolically-stressed mice. The macrophage Eh was a strong predictor of macrophage chemotaxis (r2=0.554, P<0.001). Conclusion— Thiol oxidative stress enhances macrophage recruitment into vascular and renal lesions by increasing the responsiveness of macrophages to chemoattractants. This novel mechanism contributes at least in part to accelerated atherosclerosis and kidney injury associated with dyslipidemia and diabetes in mice.

Ccl2 and Ccl3 Mediate Neutrophil Recruitment via Induction of Protein Synthesis and Generation of Lipid Mediators [Integrative Physiology/Experimental Medicine]

Objective— Although the chemokines monocyte chemoattractant protein-1 (Ccl2/JE/MCP-1) and macrophage inflammatory protein-1 (Ccl3/MIP-1) have recently been implicated in neutrophil migration, the underlying mechanisms remain largely unclear. Methods and Results— Stimulation of the mouse cremaster muscle with Ccl2/JE/MCP-1 or Ccl3/MIP-1 induced a significant increase in numbers of firmly adherent and transmigrated leukocytes (>70% neutrophils) as observed by in vivo microscopy. This increase was significantly attenuated in mice receiving an inhibitor of RNA transcription (actinomycin D) or antagonists of platelet activating factor (PAF; BN 52021) and leukotrienes (MK-886; AA-861). In contrast, leukocyte responses elicited by PAF and leukotriene-B4 (LTB4) themselves were not affected by actinomycin D, BN 52021, MK-886, or AA-861. Conversely, PAF and LTB4, but not Ccl2/JE/MCP-1 and Ccl3/MIP-1, directly activated neutrophils as indicated by shedding of CD62L and marked upregulation of CD11b. Moreover, Ccl2/JE/MCP-1- and Ccl3/MIP-1-elicited leakage of fluorescein isothiocyanate dextran as well as collagen IV remodeling within the venular basement membrane were completely absent in neutrophil-depleted mice. Conclusions— Ccl2/JE/MCP-1 and Ccl3/MIP-1 mediate firm adherence and (subsequent) transmigration of neutrophils via protein synthesis and secondary generation of leukotrienes and PAF, which in turn directly activate neutrophils. Thereby, neutrophils facilitate basement membrane remodeling and promote microvascular leakage.

Bone Marrow-Derived Cell-Specific Chemokine (C-C Motif) Receptor-2 Expression is Required for Arteriolar Remodeling [Integrative Physiology/Experimental Medicine]

Objective— Bone marrow-derived cells (BMCs) and inflammatory chemokine receptors regulate arteriogenesis and angiogenesis. Here, we tested whether arteriolar remodeling in response to an inflammatory stimulus is dependent on BMC-specific chemokine (C-C motif) receptor 2 (CCR2) expression and whether this response involves BMC transdifferentiation into smooth muscle. Methods and Results— Dorsal skinfold window chambers were implanted into C57Bl/6 wild-type (WT) mice, as well as the following bone marrow chimeras (donor-host): WT-WT, CCR2–/–-WT, WT-CCR2–/–, and EGFP+-WT. One day after implantation, tissue MCP-1 levels rose from "undetectable" to 463pg/mg, and the number of EGFP+ cells increased more than 4-fold, indicating marked inflammation. A 66% (28 µm) increase in maximum arteriolar diameter was observed over 7 days in WT-WT mice. This arteriolar remodeling response was completely abolished in CCR2–/–-WT mice but largely rescued in WT-CCR2–/– mice. EGFP+ BMCs were numerous throughout the tissue, but we found no evidence that EGFP+ BMCs transdifferentiate into smooth muscle, based on examination of >800 arterioles and venules. Conclusions— BMC-specific CCR2 expression is required for injury/inflammation-associated arteriolar remodeling, but this response is not characterized by the differentiation of BMCs into smooth muscle.

CXCR4 Expression Determines Functional Activity of Bone Marrow-Derived Mononuclear Cells for Therapeutic Neovascularization in Acute Ischemia [Integrative Physiology/Experimental Medicine]

Objective— Bone marrow–derived mononuclear cells (BMCs) improve the functional recovery after ischemia. However, BMCs comprise a heterogeneous mixture of cells, and it is not known which cell types are responsible for the induction of neovascularization after cell therapy. Because cell recruitment is critically dependent on the expression of the SDF-1-receptor CXCR4, we examined whether the expression of CXCR4 may identify a therapeutically active population of BMCs. Methods and Results— Human CXCR4+ and CXCR4– BMCs were sorted by magnetic beads. CXCR4+ BMCs showed a significantly higher invasion capacity under basal conditions and after SDF-1 stimulation. Hematopoietic or mesenchymal colony-forming capacity did not differ between CXCR4+ and CXCR4– BMCs. Injection of CXCR4+ BMCs in mice after induction of hindlimb ischemia significantly improved the recovery of perfusion compared to injection of CXCR4– BMCs. Likewise, capillary density was significantly increased in CXCR4+ BMC-treated mice. Because part of the beneficial effects of cell therapy were attributed to the release of paracrine effectors, we analyzed BMC supernatants for secreted factors. Importantly, supernatants of CXCR4+ BMCs were enriched in the proangiogenic cytokines HGF and PDGF-BB. Conclusion— CXCR4+ BMCs exhibit an increased therapeutic potential for blood flow recovery after acute ischemia. Mechanistically, their higher migratory capacity and their increased release of paracrine factors may contribute to enhanced tissue repair.

Monocyte Chemoattractant Proteins Mediate Myocardial Microvascular Dysfunction in Swine Renovascular Hypertension [Integrative Physiology/Experimental Medicine]

Background— Monocyte chemoattractant proteins (MCPs) play an important role in mediating inflammatory processes. Hypertension (HTN) is associated with inflammation as well as impaired cardiac microcirculatory function and structure, but the contribution of MCPs to these alterations remained unclear. This study tested the hypothesis that MCPs regulate cardiac microvascular function and structure in experimental HTN. Methods and Results— Pigs (n=6 per group) were studied after 10 weeks of normal, renovascular HTN, or renovascular HTN+ bindarit (MCPs inhibitor, 50 mg/kg/d PO). Left ventricular (LV) function, myocardial microvascular permeability, and fractional vascular volume were assessed by fast computed tomography before and after adenosine infusion (400 µg/kg/min). Myocardial fibrosis, inflammation, and microvascular remodeling were determined ex vivo. Hypertension was not altered by bindarit, but LV hypertrophy and diastolic function were improved. In response to adenosine, myocardial microvascular permeability increased in HTN (from 0.0083±0.0009 to 0.0103±0.0011 AU, P=0.038 versus baseline) and fractional vascular volume decreased, whereas both remained unchanged in normal and HTN+bindarit pigs. HTN upregulated endothelin-1 expression, myocardial inflammation, and microvascular wall thickening, which were inhibited by bindarit. Conclusions— MCPs partly mediate myocardial inflammation, fibrosis, vascular remodeling, and impaired vascular integrity induced by hypertension. Inhibition of MCPs could potentially be a therapeutic target in hypertensive cardiomyopathy.

Stimulation of Coronary Collateral Growth by Granulocyte Stimulating Factor: Role of Reactive Oxygen Species [Integrative Physiology/Experimental Medicine]

Objective— The purpose of this study was to determine whether G-CSF promotes coronary collateral growth (CCG) and decipher the mechanism for this stimulation. Methods and Results— In a rat model of repetitive episodic myocardial ischemia (RI, 40 seconds LAD occlusion every 20 minutes for 2 hours and 20 minutes, 3 times/d for 5 days) CCG was deduced from collateral-dependent flow (flow to LAD region during occlusion). After RI, G-CSF (100 µg/kg/d) increased CCG (P<0.01) (0.47±0.15) versus vehicle (0.14±0.06). Surprisingly, G-CSF treatment without RI increased CCG (0.57±0.18) equal to G-CSF+RI. We evaluated ROS by dihydroethidine (DHE) fluorescence (LV injection, 60 µg/kg, during two episodes of ischemia). DHE fluorescence was double in G-CSF+RI versus vehicle+RI (P<0.01), and even higher in G-CSF without RI (P<0.01). Interestingly, the DHE signal did not colocalize with myeloperoxidase (immunostaining, neutrophil marker) but appeared in cardiac myocytes. The study of isolated cardiac myocytes revealed the cytokine stimulates ROS which elicit production of angiogenic factors. Apocynin inhibited G-CSF effects both in vivo and in vitro. Conclusions— G-CSF stimulates ROS production directly in cardiomyocytes, which plays a pivotal role in triggering adaptations of the heart to ischemia including growth of the coronary collaterals.

Somitovasculin, a Novel Endothelial-Specific Transcript Involved in the Vasculature Development [Integrative Physiology/Experimental Medicine]

Objective— We recently isolated and characterized endothelial-like CD31+ cells derived from mouse embryonic stem (mES) cells and identified their transcriptome. The main objective of this study was to determine the functional relevance of the transcripts of unknown function (TUF) for vasculature development. Methods and Results— We selected 2 TUFs of more than 27 to study their role for blood vessel development in zebrafish. Morpholino (MO) knockdown of the zebrafish orthologs of the first TUF (TUF1, mouse cDNA BC022623) showed disruption of the intersegmental vessels (ISV) at 2 days postfertilization as observed by live imaging of fli:EGFP-transgenic embryos. The morphants showed abnormal blood circulation, but no effect on hematopoiesis was observed as demonstrated by gata-1 in situ hybridizations. Because knockdown of TUF1 resulted in disruption of the ISV patterning we named the TUF1 somitovasculin. TUF2 has been identified as cDNA clone BC020535. The MO knockdown of TUF2 resulted in a phenotype with an enlarged heart and the embryos lacked circulation completely. Conclusion— We have shown the participation of a novel transcript (named somitovasculin) in circulatory vessel development. The combination of expression profiling in differentiating mES cells and the zebrafish model has the potential for rapid identification and functional characterization of TUFs.

Therapeutic Potential of Unrestricted Somatic Stem Cells Isolated from Placental Cord Blood for Cardiac Repair Post Myocardial Infarction [Integrative Physiology/Experimental Medicine]

Objective— Unrestricted somatic stem cells (USSCs) were successfully identified from human cord blood. However, the efficacy of USSC transplantation for improving left ventricular (LV) function post myocardial infarction (MI) is still controversial. Methods and Results— PBS, 1x106 human fibroblasts (Fbr), 1x105 USSCs (LD), or 1x106 USSCs (HD) were transplanted intramyocardially 20 minutes after ligating the LAD of nude rats. Echocardiography and a microtip conductance catheter at day 28 revealed a dose-dependent improvement of LV function after USSC transplantation. Necropsy examination revealed dose-dependent augmentation of capillary density and inhibition of LV fibrosis. Dual-label immunohistochemistry for cardiac troponin-I and human nuclear antigen (HNA) demonstrated that human cardiomyocytes (CMCs) were dose-dependently generated in ischemic myocardium 28 days after USSC transplantation. Similarly, dual-label immunostaining for smooth muscle actin and class I human leukocyte antigen or that for von Willebrand factor and HNA also revealed a dose-dependent vasculogenesis after USSC transplantation. RT-PCR indicated that expression of human-specific genes of CMCs, smooth muscle cells, and endothelial cell markers in infarcted myocardium were significantly augmented in USSC-treated animals compared with control groups. Conclusions— USSC transplantation leads to functional improvement and recovery from MI and exhibits a significant and dose-dependent potential for concurrent cardiomyogenesis and vasculogenesis.

c-Jun DNAzymes Inhibit Myocardial Inflammation, ROS Generation, Infarct Size, and Improve Cardiac Function After Ischemia-Reperfusion Injury [Integrative Physiology/Experimental Medicine]

Objectives— Coronary reperfusion has been the mainstay therapy for reduced infarct size after a heart attack. However, this intervention also results in myocardial injury by initiating a marked inflammatory reaction, and new treatments are keenly sought. Methods and Results— The basic-region leucine zipper protein, c-Jun is poorly expressed in the normal myocardium and is induced within 24 hours after myocardial ischemia-reperfusion injury. Synthetic catalytic DNA molecules (DNAzymes) targeting c-Jun (Dz13) reduce infarct size in the area-at-risk (AAR) regardless of whether it is delivered intramyocardially at the initiation of ischemia or at the time of reperfusion. Dz13 attenuates neutrophil infiltration, c-Jun and ICAM-1 expression in vascular endothelium, cardiomyocyte apoptosis, and the generation of reactive oxygen species in the reperfused myocardium. It inhibits infiltration into the AAR of complement 3 (C3), C3a receptor (C3aR), membrane attack complex-1 (Mac-1), or matrix metalloproteinase-2 (MMP-2) positive inflammatory cells. Dz13 also improves cardiac function without influencing myocardial vascularity or fibrosis. Conclusion— These findings demonstrate the regulatory role of c-Jun in the pathogenesis of myocardial inflammation and infarction following ischemia-reperfusion injury, and inhibition of this process using catalytic DNA.

Nrf2 Protects Against Maladaptive Cardiac Responses to Hemodynamic Stress [Integrative Physiology/Experimental Medicine]

Background— Reactive oxygen species (ROS) play an important role in the maintenance of cardiovascular homeostasis. The present study sought to determine whether nuclear factor erythroid-2 related factor 2 (Nrf2), a master gene of the endogenous antioxidant defense system, is a critical regulator of the cardiac hypertrophic response to pathological stress. Methods and Results— Cardiac hypertrophy and dysfunction were established in mice by transverse aortic constriction (TAC). Nrf2 expression was transiently increased and then declined to the basal level while impairment of cardiac function proceeded. The knockout of Nrf2 (Nrf2–/–) did not cause any apparent structural and functional abnormalities in the unstressed heart. However, Nrf2–/– mice after TAC developed pathological cardiac hypertrophy, significant myocardial fibrosis and apoptosis, overt heart failure, and increased mortality, which were associated with elevated myocardial levels of 4-hydroxy-2-nonenal and 8-hydroxydeoxyguanosine and a complete blockade of the myocardial expression of several antioxidant genes. Overexpression of Nrf2 dramatically inhibited hypertrophic factor–induced ROS production and growth in both cardiomyocytes and cardiac fibroblasts, whereas knockdown of Nrf2 exerted opposite effects in both cells. Conclusions— These findings demonstrate that activation of Nrf2 provides a novel mechanism to protect the murine heart against pathological cardiac hypertrophy and heart failure via suppressing oxidative stress.

Activation of Nrf2 in Endothelial Cells Protects Arteries From Exhibiting a Proinflammatory State [Integrative Physiology/Experimental Medicine]

Objective— Proinflammatory mediators influence atherosclerosis by inducing adhesion molecules (eg, VCAM-1) on endothelial cells (ECs) via signaling intermediaries including p38 MAP kinase. Regions of arteries exposed to high shear stress are protected from inflammation and atherosclerosis, whereas low-shear regions are susceptible. Here we investigated whether the transcription factor Nrf2 regulates EC activation in arteries. Methods and Results— En face staining revealed that Nrf2 was activated in ECs at an atheroprotected region of the murine aorta where it negatively regulated p38–VCAM-1 signaling, but was expressed in an inactive form in ECs at an atherosusceptible site. Treatment with sulforaphane, a dietary antioxidant, activated Nrf2 and suppressed p38–VCAM-1 signaling at the susceptible site in wild-type but not Nrf2–/– animals, indicating that it suppresses EC activation via Nrf2. Studies of cultured ECs revealed that Nrf2 inactivates p38 by suppressing an upstream activator MKK3/6 and by enhancing the activity of the negative regulator MKP-1. Conclusions— Nrf2 prevents ECs at the atheroprotected site from exhibiting a proinflammatory state via the suppression of p38–VCAM-1 signaling. Pharmacological activation of Nrf2 reduces EC activation at atherosusceptible sites and may provide a novel therapeutic strategy to prevent or reduce atherosclerosis.

Dynamic Observation of Mechanically-Injured Mouse Femoral Artery Reveals an Antiinflammatory Effect of Renin Inhibitor [Integrative Physiology/Experimental Medicine]

Objective— The renin-angiotensin-aldosterone system (RAS) plays a central role in atherosclerosis. To investigate the effects of a direct renin inhibitor aliskiren on vascular inflammation, we conducted leukocyte adhesion assays in vivo and in vitro using a novel real-time imaging system. Methods and Results— Aliskiren (10 mg/kg/d) or PBS was administered to C57BL/6 mice (6–7 weeks of age; Oriental Yeast, Tokyo, Japan) for 2 weeks via an osmotic pump. Blood pressure was not significantly changed in the 2 groups throughout the experimental period. A perivascular cuff injury was then introduced to the femoral artery and real-time intravital microscopic observation was conducted 24 hours after injury. The number of adherent leukocytes was elevated in the injured mice without aliskiren (43.8±9.3/10–2 mm2), whereas that was significantly reduced in the mice with aliskiren (18.4±4.4, P<0.05). Treatment of human umbilical vein endothelial cells (HUVECs) with aliskiren significantly reduced the adhesion of THP-1 cells to TNF-–activated HUVECs (P<0.05). Interestingly, TNF-–induced renin activity and angiotensin II production in HUVECs were also blunted by aliskiren. Furthermore, exogenous renin and angiotensin II abrogated the aliskiren-mediated reduction of THP-1 cell adhesion to HUVECs. Conclusions— Our in vivo and in vitro findings indicate a pivotal role for renin inhibition in vascular inflammation independent of blood pressure.

The {alpha}11{beta}1 Integrin Has a Mechanistic Role in Control of Interstitial Fluid Pressure and Edema Formation in Inflammation [Integrative Physiology/Experimental Medicine]

Objective— Collagen-binding integrins may be involved in controlling interstitial fluid pressure (Pif), transcapillary fluid flux, and tissue fluid volume. Our aim was to explore whether the newly discovered collagen binding 11β1 integrin has a mechanistic role in inflammatory edema formation. Methods and Results— In collagen matrices seeded with a mixture of mast cells and fibroblasts, fibroblasts lacking the 11 integrin subunit (11–/–) contracted collagen gels less efficiently than control fibroblasts, suggesting that the 11β1 integrin is able to mediate tensile force in connective tissues. In 11–/– mice, control Pif in skin did not differ from the pressure found in wild-type mice. Whereas a reduction in Pif was found in control mice after inducing inflammation, thereby contributing to fluid extravasation and edema formation, such a reduction was not seen in 11–/– mice. That this effect is mediated through the extracellular compartment is suggested by a similar plasma protein extravasation ratio in 11–/– and wild-type mice. Conclusions— Our data suggest that 11β1 integrins on dermal fibroblasts mediate collagen lattice remodeling and have a mechanistic role in controlling Pif in inflammation and thereby fluid extravasation and edema formation in vivo.

PPAR{beta}/{delta} Agonists Modulate Platelet Function via a Mechanism Involving PPAR Receptors and Specific Association/Repression of PKC{alpha}-Brief Report [Cell Biology/Signaling]

Objectives— Peroxisome proliferator-activated receptor β/ (PPARβ/) is a nuclear receptor found in platelets. PPARβ/ agonists acutely inhibit platelet function within a few minutes of addition. As platelets are anucleated, the effects of PPARβ/ agonists on platelets must be nongenomic. Currently, the particular role of PPARβ/ receptors and their intracellular signaling pathways in platelets are not known. Methods and Results— We have used mice lacking PPARβ/ (PPARβ/–/–) to show the effects of the PPARβ/ agonist GW501516 on platelet adhesion and cAMP levels are mediated specifically by PPARβ/, however GW501516 had no PPARβ/-specific effect on platelet aggregation. Studies in human platelets showed that PKC, which can mediate platelet activation, was bound and repressed by PPARβ/ after platelets were treated with GW501516. Conclusions— These data provide evidence of a novel mechanism by which PPAR receptors influence platelet activity and thereby thrombotic risk.

Foxp3 Regulates Megakaryopoiesis and Platelet Function [Cell Biology/Signaling]

Objective— Platelets are crucial for hemostasis and are vital regulators of inflammation. Foxp3 is a key transcription factor for T regulatory cell development. Humans with IPEX (immune dysregulation, polyendocrinopathy, enteropathy, x-linked) and the scurfy (Foxp3sf) mouse have mutations in the Foxp3 gene that lead to a host of pathologies including autoimmunity and skin diseases. Scurfy mice and some humans with IPEX are also thrombocytopenic. The purpose of this study was to determine whether the absence of functional Foxp3 leads to defects in megakaryocytes and platelets. Methods and Results— We discovered that human and mouse megakaryocytes express Foxp3 mRNA and protein. Using shRNA and Foxp3sf mice, we demonstrated that Foxp3-deficient mouse and human megakaryocyte progenitors exhibited proliferation defects. Striking platelet abnormalities were observed in both an IPEX patient and Foxp3sf mice. Impaired platelet spreading and release of TGF-β and CD40 ligand (CD40L), and abnormal levels of plasma CD40L were observed in a case of IPEX syndrome. Foxp3sf mice were thrombocytopenic and had increased platelet volume and altered serum levels of CD40L, TXB2, and TGF-β. Conclusion— These findings provide compelling new evidence that Foxp3 is needed for proper megakaryopoiesis and plays a role in regulating platelet function including spreading and release.

Rapid Procoagulant Phosphatidylserine Exposure Relies on High Cytosolic Calcium Rather Than on Mitochondrial Depolarization [Cell Biology/Signaling]

Objective— Relationships between intracellular Ca2+ concentration ([Ca2+]cyt) and apoptotic events, such as mitochondrial depolarization (m loss) and Bcl-2 and Bad phosphorylation, were analyzed in platelets and Jurkat cells in relation to rapid procoagulant phosphatidylserine (PS) exposure. Methods and Results— Platelets were stimulated with A23187, thapsigargin (TG) and thrombin plus convulxin (Thr/Cvx), and Jurkat cells with ionomycin, in the presence or absence of cyclosporin A (CsA), a mitochondrial permeability transition pore inhibitor. m loss occurred when platelets were stimulated in Ca2+ medium in conditions exposing PS, but also in EGTA medium. CsA inhibited PS exposure, [Ca2+]cyt increase, and m loss in platelets stimulated with TG and Thr/Cvx, but had no inhibitory effect on A23187 stimulation. CsA reduced TG-induced Ca2+ release from the endoplasmic reticulum and, consequently, external Ca2+ influx. In ionomycin-stimulated Jurkat cells, rapid PS exposure was evidenced but not m loss, and CsA did not inhibit the process. The status of phosphorylated Bad and Bcl-2 in both cell types remained unchanged on stimulation. Conclusions— Whether m loss occurs or not, PS exposure is triggered by a high [Ca2+]cyt increase. Data further demonstrate that CsA prevents membrane scrambling by inhibiting the high [Ca2+]cyt increase, independently of its effect on mitochondrial permeability transition pore.

Disinhibition of SOD-2 Expression to Compensate for a Genetically Determined NO Deficit in Endothelial Cells-Brief Report [Cell Biology/Signaling]

Objective— Homozygosity for the –786C-variant of the human nos-3 gene is a risk factor for coronary artery disease (CAD). Interestingly, affected individuals develop CAD more frequently but not earlier than the general population. Methods and Results— Genotyped primary human umbilical vein endothelial cells (ECs) were exposed to fluid shear stress (FSS) and analyzed for nitric oxide (NO) and superoxide anion (O2–) formation as well as mRNA and protein expression of different antioxidant enzymes. Dysfunctional CC-genotype ECs failed to upregulate NO synthase expression in response to FSS and exhibited a reduced NO synthesis capacity when compared to functionally intact TT-genotype ECs. However, only CC-genotype ECs responded to FSS with an Egr-1–mediated increase in manganese-containing superoxide dismutase (SOD-2) expression, shielding them from endothelin-1–induced oxidative stress in a NO-independent manner. Conclusions— This FSS-induced rise in SOD-2 expression in CC-genotype ECs effectively stabilizes their antiatherosclerotic phenotype and may explain not only the comparatively slow onset of CAD in homozygous carriers of the C-allele of the nos-3 gene but also define a general strategy for preventing endothelial dysfunction at the outset of atherosclerosis.

Upregulation of Glutathione Peroxidase Offsets Stretch-Induced Proatherogenic Gene Expression in Human Endothelial Cells [Cell Biology/Signaling]

Objective— Localization of atherosclerotic plaques typically correlates with areas of biomechanical strain where shear stress is decreased while stretch, thought to promote atherogenesis through enhanced oxidative stress, is increased. Methods and Results— In human cultured endothelial cells, nitric oxide synthase expression was exclusively shear stress–dependent whereas expression of glutathione peroxidase-1 (GPx-1), but not that of Cu2+/Zn2+-superoxide dismutase or Mn2+-superoxide dismutase, was upregulated solely in response to cyclic stretch. GPx-1 expression was also enhanced in isolated mouse arteries perfused at high pressure. Combined pharmacological and decoy oligodeoxynucleotide blockade revealed that activation of p38 MAP kinase followed by nuclear translocation of CCAAT/enhancer binding protein plays a pivotal role in stretch-induced GPx-1 expression in human endothelial cells. Antisense oligodeoxynucleotide knockdown of GPx-1 reinforced both their capacity to generate hydrogen peroxide and the transient stretch-induced expression of CD40, monocyte chemoatractant protein-1, and vascular cell adhesion molecule-1. Consequently, THP-1 monocyte adhesion to the GPx-1–depleted cells was augmented. Conclusions— Stretch-induced proatherosclerotic gene expression in human endothelial cells seems to be hydrogen peroxide-mediated. The concomitant rise in GPx-1 expression, but not that of other antioxidant enzymes, may comprise an adaptive mechanism through which the cells maintain their antiatherosclerotic properties in spite of a decreased bioavailability of nitric oxide.

Flow Activation of AMP-Activated Protein Kinase in Vascular Endothelium Leads to Kruppel-Like Factor 2 Expression [Cell Biology/Signaling]

Objective— Vascular endothelial cells (ECs) confer atheroprotection at locations of the arterial tree where pulsatile laminar flow (PS) exists with a high shear stress and a large net forward direction. We investigated whether the PS-induced expression of the transcription factor Krüppel-Like Factor 2 (KLF2) in cultured ECs and its expression in the mouse aorta is regulated by AMP-activated protein kinase (AMPK). Methods and Results— AMPK inhibition by Compound C or siRNA had a significant blocking effect on the PS-induced KLF2 expression. The induction of KLF2 by PS led to the increase in eNOS and the suppression of ET-1, which could be reversed by KLF2 siRNA. In addition, PS induced the phosphorylation of ERK5 and MEF2 which are necessary for the KLF2 expression. These mechanotransduction events were abrogated by the blockade of AMPK. Furthermore, the phosphorylation levels of ERK5 and MEF2, as well as the expression of KLF2, were significantly reduced in the aorta of AMPK2 knockout mice when compared with wild-type control mice. Conclusion— The flow-mediated AMPK activation is a newly defined KLF2 regulatory pathway in vascular endothelium that acts via ERK5/MEF2.

Alzheimer Disease-Associated Peptide, Amyloid {beta}40, Inhibits Vascular Regeneration With Induction of Endothelial Autophagy [Cell Biology/Signaling]

Objective— Although the majority of cases of Alzheimer disease (AD) are known to be attributable to the sporadic (nongenetic) form of the disease, the mechanism underlying its cause and progression still remains unclear. Methods and Results— We found that vascular β-amyloid (Aβ), Aβ40, inhibited the proliferative activity of human brain vascular endothelial cells (HBECs) without toxic effects on them. This peptide also inhibited tube formation and migration of HBECs. Moreover, Aβ40 inhibited ex vivo hippocampal revascularization, reendothelialization, and the differentiation of adult endothelial progenitor cells. Importantly, Aβ40 suppressed the proliferative activity of HBECs through the induction of "self-digesting" autophagy. This induction involved the intracellular regulation of class 3 phosphatidylinositol 3-kinase (PI3K) as well as Akt signaling in HBECs. Furthermore, tissue culture of murine brain sections from GFP-LC3 transgenic mice revealed that Aβ40 not only reduced the vessel density in hippocampal lesions, but also induced autophagy in neurovascular ECs. Conclusions— Our present findings indicate that the initial progression of AD might be in part driven by Aβ40-induced endothelial autophagy and impairment of neurovascular regeneration, suggesting important implications for therapeutic approaches to AD.

The Phosphorylation Motif at Serine 225 Governs the Localization and Function of Sphingosine Kinase 1 in Resistance Arteries [Cell Biology/Signaling]

Objective— The purpose of this study was to characterize a phosphorylation motif at serine 225 as a molecular switch that regulates the pressure-dependent activation of sphingosine kinase 1 (Sk1) in resistance artery smooth muscle cells. Methods and Results— In isolated hamster gracilis muscle resistance arteries, pressure-dependent activation/translocation of Sk1 by ERK1/2 was critically dependent on its serine 225 phosphorylation site. Specifically, expression of Sk1S225A reduced resting and myogenic tone, resting Ca2+, pressure-induced Ca2+ elevations, and Ca2+ sensitivity. The lack of function of the Sk1S225A mutant could not be entirely overcome by forced localization to the plasma membrane via a myristoylation/palmitylation motif; the membrane anchor also significantly inhibited the function of the wild-type Sk1 enzyme. In both cases, Ca2+ sensitivity and myogenic tone were attenuated, whereas Ca2+ handling was normalized/enhanced. These discrete effects are consistent with cell surface receptor-mediated effects (Ca2+ sensitivity) and intracellular effects of S1P (Ca2+ handling). Accordingly, S1P2 receptor inhibition (1µmol/L JTE013) attenuated myogenic tone without effect on Ca2+. Conclusions— Translocation and precise subcellular positioning of Sk1 is essential for full Sk1 function; and two distinct S1P pools, proposed to be intra- and extracellular, contribute to the maintenance of vascular tone.

Liver X Receptor-Mediated Induction of Cholesteryl Ester Transfer Protein Expression Is Selectively Impaired in Inflammatory Macrophages [Cell Biology/Signaling]

Objective— Cholesteryl ester transfer protein (CETP) is a target gene for the liver X receptor (LXR). The aim of this study was to further explore this regulation in the monocyte-macrophage lineage and its modulation by lipid loading and inflammation, which are key steps in the process of atherogenesis. Methods and Results— Exposure of bone marrow–derived macrophages from human CETP transgenic mice to the T0901317 LXR agonist increased CETP, PLTP, and ABCA1 mRNA levels. T0901317 also markedly increased CETP mRNA levels and CETP production in human differentiated macrophages, whereas it had no effect on CETP expression in human peripheral blood monocytes. In inflammatory mouse and human macrophages, LXR-mediated CETP gene upregulation was inhibited, even though ABCA1, ABCG1, and SREBP1c inductions were maintained. The inhibition of CETP gene response to LXR agonists in inflammatory cells was independent of lipid loading (ie, oxidized LDL increased CETP production in noninflammatory macrophages with a synergistic effect of synthetic LXR agonists). Conclusion— LXR-mediated induction of human CETP expression is switched on during monocyte-to-macrophage differentiation, is magnified by lipid loading, and is selectively lost in inflammatory macrophages, which suggests that inflammatory cells may not increase the circulating CETP pool on LXR agonist treatment.

Stimulation of Cholesterol Efflux by LXR Agonists in Cholesterol-Loaded Human Macrophages Is ABCA1-Dependent but ABCG1-Independent [Cell Biology/Signaling]

Objective— Maintenance of cholesterol homeostasis in human macrophages is essential to prevent foam cell formation. We evaluated the relative contribution of the ABCA1 and ABCG1 transporters to cholesterol efflux from human macrophages, and of the capacity of LXR agonists to reduce foam cell formation by stimulating export of cellular cholesterol. Methods and Results— ABCG1 mRNA levels were strongly increased in acLDL-loaded THP-1 macrophages and in HMDM on stimulation with LXR agonists. However, silencing of ABCG1 expression using ABCG1-specific siRNA indicated that ABCG1 was not essential for cholesterol efflux to HDL in cholesterol-loaded human macrophages stimulated with LXR agonists. Indeed, ABCA1 was solely responsible for the stimulation of cholesterol efflux to HDL on LXR activation, as this effect was abolished in HMDM from Tangier patients. Furthermore, depletion of cellular ATP indicated that the LXR-induced export of cholesterol was an ATP-dependent transport mechanism in human macrophages. Finally, use of an anti–Cla-1 blocking antibody identified the Cla-1 receptor as a key component in cholesterol efflux to HDL from cholesterol-loaded human macrophages. Conclusion— Our data indicate that stimulation of cholesterol efflux to HDL by LXR agonists in human foam cells involves an ATP-dependent transport mechanism mediated by ABCA1 that it appears to be independent of ABCG1 expression.

Inhibition of Long-Chain Acyl Coenzyme A Synthetases During Fatty Acid Loading Induces Lipotoxicity in Macrophages [Cell Biology/Signaling]

Objectives— Obesity is often associated with hypertriglyceridemia and elevated free fatty acids (FFAs), which are independent risk factors for cardiovascular disease and diabetes. Although impairment of cholesterol homeostasis is known to induce toxicity in macrophages, the consequence of altered fatty acid homeostasis is not clear. Methods and Results— Long-chain acyl CoA synthetases (ACSLs) play a critical role in fatty acid homeostasis by channeling fatty acids to diverse metabolic pools. We treated mouse peritoneal macrophages (MPMs) with VLDL or FFAs in the presence of triacsin C, an inhibitor of the 3 ACSL isoforms present in macrophages. Treatment of macrophages with VLDL and triacsin C resulted in reduced TG accumulation but increased intracellular FFA levels, which induced lipotoxicity characterized by apoptosis. Treatment of MPMs with the saturated fatty acid stearic acid in the presence of triacsin C increased intracellular stearic acid and induced apoptosis. Stromal vascular cells collected from high-fat diet–fed mice displayed foam cell morphology and exhibited increased mRNA levels of macrophage markers and ACSL1. Importantly, all of these changes were associated with increased FFA level in AT. Conclusions— Inhibition of ACSLs during fatty acid loading results in apoptosis via accumulation of FFAs. Our data have implications in understanding the consequences of dysregulated fatty acid metabolism in macrophages.

Saturated Fatty Acids Do Not Directly Stimulate Toll-Like Receptor Signaling [Cell Biology/Signaling]

Objective— Toll-like receptors (TLRs) initiate inflammatory signaling in response to conserved microbial molecules. It has been proposed that dietary saturated fatty acids (SFAs) may also serve as endogenous ligands of TLR2 or TLR4, thereby promoting diseases associated with inflammation and dyslipidemia, including atherosclerosis and insulin resistance. Methods and Results— We investigated the effects of SFAs on TLR-dependent signaling using a broad range of cell types and readouts. In HEK-293 cells transfected with TLR2, TLR4, or TLR5, SFAs complexed with fatty-acid-free bovine serum albumin (BSA)-stimulated TLR-dependent signaling. However, SFAs alone did not elicit a similar response. Further analysis showed that the effect seen with the complexed SFAs was attributable to LPS and lipopeptide contamination of fatty-acid-free BSA. Additional studies in macrophages, endothelial cells, smooth muscle cells, adipocytes, skeletal muscle cells, and human peripheral blood mononuclear cells confirmed the lack of stimulation of TLR-dependent signaling pathways or expression of TLR-target genes by SFAs. Conclusions— SFAs do not directly stimulate TLR-dependent signaling, suggesting that alternative mechanisms link dietary fat intake with TLR-associated pathologies. LPS and lipopeptide contamination of the widely used reagent fatty-acid-free BSA explains the previously reported stimulation of TLR2 and TLR4 by SFAs.

Statins Block Calcific Nodule Formation of Valvular Interstitial Cells by Inhibiting {alpha}-Smooth Muscle Actin Expression [Cell Biology/Signaling]

Objective— Calcific aortic stenosis, characterized by excessive fibrosis and deposition of bone-like calcified tissue, affects roughly 2% to 3% of the U.S. population over the age of 65. Recent studies have suggested that statins have a positive effect on the progression of aoritic stenosis, likely because of their ability to affect the resident cell population, known as valvular interstitial cells (VICs). VICs are fibroblastic cells that can differentiate to form activated myofibroblasts, displaying increased alpha smooth muscle actin (SMA) expression, contractility, and collagen production. Methods and Results— In culture, VICs spontaneously form multicellular aggregates that subsequently develop into calcified nodules, providing an in vitro model for aortic stenosis. Using real-time microscopic tracking, we observed that confluent VIC monolayers spontaneously contract into rounded nodules, suggesting that myofibroblastic contractility is a critical step in the process of nodule formation. Overexpression of SMA increased VIC calcific nodule formation and contractility, whereas knockdown of SMA with siRNAs reduced these phenotypes, suggesting that the expression and contractile properties of SMA are essential to the formation of nodules. Statin treatment of VICs reduced SMA expression, inhibited contractility, and decreased nodule formation. When statins were used to treat preformed nodules, no decrease in the number of calcified nodules was observed, suggesting that statins may play more of a preventative role in aortic stenosis than a cure. Conclusions— Our studies provide evidence of a causal relationship between VIC myofibroblastic activity and initial VIC calcific nodule formation. Furthermore, we demonstrate that pravastatin inhibition of calcific nodule formation is related to inhibition of myofibroblastic activity.

Genome-Wide Association Identifies the ABO Blood Group as a Major Locus Associated With Serum Levels of Soluble E-Selectin [Clinical and Population Studies]

Background— Elevated serum soluble E-selectin levels have been associated with a number of diseases. Although E-selectin levels are heritable, little is known about the specific genetic factors involved. E-selectin levels have been associated with the ABO blood group phenotype. Methods and Results— We performed a high-resolution genome-wide association study of serum soluble E-selectin levels in 685 white individuals with type 1 diabetes from the Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Intervention and Complications (EDIC) study to identify major loci influencing levels. Highly significant evidence for association (P=10–29) was observed for rs579459 near the ABO blood group gene, accounting for 19% of the variance in E-selectin levels. Levels of E-selectin were higher in O/O than O/A heterozygotes, which were likewise higher than A/A genotypes. Analysis of subgroups of A alleles reveals heterogeneity in the association, and even after this was accounted for, an intron 1 SNP remained significantly associated. We replicate the ABO association in nondiabetic individuals. Conclusion— ABO is a major locus for serum soluble E-selectin levels. We excluded population stratification, fine-mapped the association to sub-A alleles, and also document association with additional variation in the ABO region.

FVII, FVIIa, and Downstream Markers of Extrinsic Pathway Activation Differ by EPCR Ser219Gly Variant in Healthy Men [Clinical and Population Studies]

Objective— The purpose of this study was to determine the effect of a variant in EPCR (Ser219Gly), previously shown to affect EPCR shedding, on plasma FVII, FVIIa, and downstream markers of activated coagulation. Methods and Results— Statistical analysis was undertaken in 2000 healthy middle aged men (NPHSII). Higher soluble EPCR levels were confirmed for Gly allele carriers (P<0.0001). Significantly higher levels of FVII, FVIIa, and downstream markers of activated coagulation in the extrinsic pathway (FIX activation pep [FIXpep]; FX activation pep [FXpep]), and prothrombin F1+2 (F1+2) were identified in baseline samples, in Gly carriers compared to Ser/Ser (P<=0.04 for trend). In repeat samples collected for up to 5 years, levels of FVII and F1+2 were higher in Gly allele carriers compared to Ser/Ser by (FVII: 6.9% CI 5.5 to 8.4 in Ser/Gly; and 23.4% CI 16.3 to 30.8 in Gly/Gly, P<0.0001), (F1+2: 8.1% CI 5.2 to 11.1 in Ser/Gly; 25.2% CI 11.8 to 40.3 in Gly/Gly, P<0.04), confirming reproducibility of findings at baseline. Molar ratios for FIXpep, FXpep, and F1+2 to FVIIa were constant in Ser/Ser and Ser/Gly but tended to be higher in Gly/Gly, reaching statistical significance for FIXpep:FVIIa (P=0.04). Conclusions— These data suggest that higher levels of FVII and FVIIa circulate when EPCR shedding is greatest. Furthermore, these results suggest consequences for activation of extrinsic coagulation.

Ion Mobility Analysis of Lipoprotein Subfractions Identifies Three Independent Axes of Cardiovascular Risk [Clinical and Population Studies]

Objective— Whereas epidemiological studies show that levels of low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) predict incident cardiovascular disease (CVD), there is limited evidence relating lipoprotein subfractions and composite measures of subfractions to risk for CVD in prospective cohort studies. Methods and Results— We tested whether combinations of lipoprotein subfractions independently predict CVD in a prospective cohort of 4594 initially healthy men and women (the Malmö Diet and Cancer Study, mean follow-up 12.2 years, 377 incident cardiovascular events). Plasma lipoproteins and lipoprotein subfractions were measured at baseline with a novel high-resolution ion mobility technique. Principal component analysis (PCA) of subfraction concentrations identified 3 major independent (ie, zero correlation) components of CVD risk, one representing LDL-associated risk, a second representing HDL-associated protection, and the third representing a pattern of decreased large HDL, increased small/medium LDL, and increased triglycerides. The last corresponds to the previously described "atherogenic lipoprotein phenotype." Several genes that may underlie this phenotype—CETP, LIPC, GALNT2, MLXIPL, APOA1/A5, LPL—are suggested by SNPs associated with the combination of small/medium LDL and large HDL. Conclusion— PCA on lipoprotein subfractions yielded three independent components of CVD risk. Genetic analyses suggest these components represent independent mechanistic pathways for development of CVD.