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[2009 ATVB Abstracts] Arteriosclerosis, Thrombosis, and Vascular Biology Annual Conference 2009

[Brief Reviews] Cardiovascular Molecular Imaging

[Brief Reviews] Molecular Imaging in Atherosclerosis, Thrombosis, and Vascular Inflammation

Appreciation of the molecular and cellular processes of atherosclerosis, thrombosis, and vascular inflammation has identified new targets for imaging. The common goals of molecular imaging approaches are to accelerate and refine diagnosis, provide insights that reveal disease diversity, guide specific therapies, and monitor the effects of those therapies. Here we undertake a comparative analysis of imaging modalities that have been used in this disease area. We consider the elements of contrast agents, emphasizing how an understanding of the biology of atherosclerosis and its complications can inform optimal design. We address the potential and limitations of current contrast approaches in respect of translation to clinically usable agents and speculate on future applications.

[Brief Reviews] Nanotechnology in Medical Imaging: Probe Design and Applications

Nanoparticles have become more and more prevalent in reports of novel contrast agents, especially for molecular imaging, the detection of cellular processes. The advantages of nanoparticles include their potency to generate contrast, the ease of integrating multiple properties, lengthy circulation times, and the possibility to include high payloads. As the chemistry of nanoparticles has improved over the past years, more sophisticated examples of nano-sized contrast agents have been reported, such as paramagnetic, macrophage targeted quantum dots or vβ3-targeted, MRI visible microemulsions that also carry a drug to suppress angiogenesis. The use of these particles is producing greater knowledge of disease processes and the effects of therapy. Along with their excellent properties, nanoparticles may produce significant toxicity, which must be minimized for (clinical) application. In this review we discuss the different factors that are considered when designing a nanoparticle probe and highlight some of the most advanced examples.

[Brief Reviews] Iron Oxide Particles for Atheroma Imaging

The selection of patients for vascular interventions has been solely based on luminal stenosis and symptomatology. However, histological data from both the coronary and carotid vasculature suggest that other plaque features such as inflammation may be more important in predicting future thromboembolic events. Ultrasmall superparamagnetic iron oxide (USPIO) contrast agents have been used for noninvasive MRI assessment of atherosclerotic plaque inflammation in humans. It has reached the stage of development to have been recently used in an interventional drug study to not only assess inflammatory progression but also select patients at high risk. This article reviews the basic science behind the use of USPIO contrast agents in atheroma MR imaging, experimental work in animals, and how this has led to the emergence of this promising targeted imaging platform for assessment of high risk carotid atherosclerosis in humans.

[Brief Reviews] Inflammation Imaging in Atherosclerosis

Inflammation is important at many stages of atherosclerotic plaque development. We highlight several imaging modalities that can quantify the degree of plaque inflammation noninvasively. Imaging of this type might allow testing of novel antiatherosclerosis drugs, identification of patients at risk of plaque rupture, and deeper insight into the biology of the disease. The imaging modalities are discussed in relation to their potential use in these areas.

[Brief Reviews] Optical and Multimodality Molecular Imaging: Insights Into Atherosclerosis

Imaging approaches that visualize molecular targets rather than anatomic structures aim to illuminate vital molecular and cellular aspects of atherosclerosis biology in vivo. Several such molecular imaging strategies stand ready for rapid clinical application. This review describes the growing role of in vivo optical molecular imaging in atherosclerosis and highlights its ability to visualize atheroma inflammation, calcification, and angiogenesis. In addition, we discuss advances in multimodality probes, both in the context of multimodal imaging as well as multifunctional, or "theranostic," nanoparticles. This review highlights particular molecular imaging strategies that possess strong potential for clinical translation.

[Brief Reviews] In Vivo Imaging of Stem Cells and Beta Cells Using Direct Cell Labeling and Reporter Gene Methods

Cellular transplantation therapy offers a means to stimulate cardiovascular repair either by direct (graft-induced) or indirect (host-induced) tissue regeneration or angiogenesis. Typically, autologous or donor cells of specific subpopulations are expanded exogenously before administration to enrich the cells most likely to participate in tissue repair. In animal models of cardiovascular disease, the fate of these exogenous cells can be determined using histopathology. Recently, methods to label cells with contrast agents or transduce cells with reporter genes to produce imaging beacons has enabled the serial and dynamic assessment of the survival, fate, and engraftment of these cells with noninvasive imaging. Although cell tracking methods for cardiovascular applications have been most studied in stem or progenitor cells, research in tracking of whole islet transplants and particularly insulin producing beta cells has implications to the cardiovascular community attributable to the vascular changes associated with diabetes mellitus. In this review article, we will explore some of the state-of-the art methods for stem, progenitor, and beta cell tracking.

[Brief Reviews] Molecular Imaging of Macrophage Cell Death for the Assessment of Plaque Vulnerability

The ability to identify atherosclerotic plaques that are prone to rupture, also called vulnerable plaques, may provide a major step forward in the recognition of patients that have a high risk of developing acute myocardial infarction. Current clinical risk profiling algorithms, such as the Framingham and Procam risk scores, have reasonable predictive value in the assessment of the 10 year risk. These clinical risk profiling scores typically classify patients into low risk (10-year risk, less than 5%), intermediate risk (5% to 20% risk), and high risk (greater than 20%). The challenge to imagers is to identify the risk that is beyond 2% yearly risk. Molecular imaging may help identify plaque inflammation and apoptosis of inflammatory cells, which are obligatory components of the plaque instability. These processes offer specific biological targets that can potentially be exploited to obtain biological information on atherosclerosis development in the individual patient.

[Integrative Physiology/Experimental Medicine] Disruption of SEMA4D Ameliorates Platelet Hypersensitivity in Dyslipidemia and Confers Protection Against the Development of Atherosclerosis

Objective— In dyslipidemic states, platelets become hyperreactive, secreting molecules that promote atherosclerosis. We have shown that the semaphorin family member, sema4D (CD100), is expressed on the surface of platelets and proposed that its role includes promoting thrombus growth by binding to nearby platelets and endothelial cells, both of which express sema4D receptors. Here we tested the hypothesis that deleting sema4D will attenuate the adverse consequences of dyslipidemia on platelets and the vessel wall. Methods and Results— Platelet function and atherosclerotic lesion formation were measured in LDLR(–/–) and sema4D(–/–)LDLR(–/–) mice after 6 months on a high-fat diet. All of the mice developed the dyslipidemia expected on this diet in the absence of functional LDL receptors. However, when compared to LDLR(–/–) mice, sema4D(–/–) LDLR(–/–) mice had reduced lipid deposition in the descending aorta, a 6-fold decrease in the frequency of arterial occlusion and a reduction to near wild-type levels in the accumulation of platelets after injury. These differences were retained ex vivo, with a marked decrease in platelet accumulation on collagen under flow and in platelet aggregation. Conclusions— These results show that loss of sema4D expression reduces the platelet hyperactivity otherwise found in dyslipidemia, and confers protection against the development of atherosclerosis.

[Integrative Physiology/Experimental Medicine] Inactivation of the Adenosine A2A Receptor Protects Apolipoprotein E-Deficient Mice From Atherosclerosis

Background— Atherosclerosis is a chronic inflammatory disease of the arterial vessel wall. The A2A receptor (A2AR) plays a central role in many antiinflammatory effects of adenosine. However, the role of A2AR in atherosclerosis is not clear. Methods and Results— The knockout of A2AR in apolipoprotein E–deficient (Apoe–/–/A2AR–/–) mice led to an increase in body weight and levels of blood cholesterol and proinflammatory cytokines, as well as the inflammation status of atherosclerotic lesions. Unexpectedly, Apoe–/–/A2AR–/– mice developed smaller lesions, as did chimeric Apoe–/– mice lacking A2AR in bone marrow–derived cells (BMDCs). The lesions of those mice exhibited a low density of foam cells and the homing ability of A2AR-deficient monocytes did not change. Increased foam cell apoptosis was detected in atherosclerotic lesions of Apoe–/–/A2AR–/– mice. In the absence of A2AR, macrophages incubated with oxidized LDL or in vivo–formed foam cells also exhibited increased apoptosis. A2AR deficiency in foam cells resulted in an increase in p38 mitogen–activated protein kinase (MAPK) activity. Inhibition of p38 phosphorylation abrogated the increased apoptosis of A2AR-deficient foam cells. Conclusion— Inactivation of A2AR, especially in BMDCs, inhibits the formation of atherosclerotic leisons, suggesting that A2AR inactivation may be useful for the treatment of atherosclerosis.

[Integrative Physiology/Experimental Medicine] Core2 1-6-N-Glucosaminyltransferase-I Deficiency Protects Injured Arteries From Neointima Formation in ApoE-Deficient Mice

Objective— Core2 1 to 6-N-glucosaminyltransferase-I (C2GlcNAcT-I) plays an important role in optimizing the binding functions of several selectin ligands, including P-selectin glycoprotein ligand. We used apolipoprotein E (ApoE)-deficient atherosclerotic mice to investigate the role of C2GlcNAcT-I in platelet and leukocyte interactions with injured arterial walls, in endothelial regeneration at injured sites, and in the formation of arterial neointima. Methods and Results— Arterial neointima induced by wire injury was smaller in C2GlcNAcT-I-deficient apoE–/– mice than in control apoE–/– mice (a 79% reduction in size). Compared to controls, apoE–/– mice deficient in C2GlcNAcT-I also demonstrated less leukocyte adhesion on activated platelets in microflow chambers (a 75% reduction), and accumulation of leukocytes at injured areas of mouse carotid arteries was eliminated. Additionally, endothelial regeneration in injured lumenal areas was substantially faster in C2GlcNAcT-I-deficient apoE–/– mice than in control apoE–/– mice. Endothelial regeneration was associated with reduced accumulation of platelet factor 4 (PF4) at injured sites. PF4 deficiency accelerated endothelial regeneration and protected mice from neointima formation after arterial injury. Conclusions— C2GlcNAcT-I deficiency suppresses injury-induced arterial neointima formation, and this effect is attributable to decreased leukocyte recruitment to injured vascular walls and increased endothelial regeneration. Both C2GlcNAcT-I and PF4 are promising targets for the treatment of arterial restenosis.

[Integrative Physiology/Experimental Medicine] Insulin Increases Reendothelialization and Inhibits Cell Migration and Neointimal Growth After Arterial Injury

Objective— Insulin has both growth-promoting and protective vascular effects in vitro, however the predominant effect in vivo is unclear. We investigated the effects of insulin in vivo on neointimal growth after arterial injury. Methods and Results— Rats were given subcutaneous control (C) or insulin implants (3U/d;I) 3 days before arterial (carotid or aortic) balloon catheter injury. Normoglycemia was maintained by oral glucose and, after surgery, by intraperitoneal glucose infusion (saline in C). Insulin decreased intimal area (P<0.01) but did not change intimal cell proliferation or apoptosis. However, insulin inhibited cell migration into the intima (P<0.01) and increased expression of smooth muscle cell (SMC) differentiation markers (P<0.05). Insulin also increased reendothelialization (P<0.01) and the number of circulating progenitor cells (P<0.05). Conclusions— These results are the first demonstration that insulin has a protective effect on both SMC and endothelium in vivo, resulting in inhibition of neointimal growth after vessel injury.

[Integrative Physiology/Experimental Medicine] PECAM-1 Is Necessary for Flow-Induced Vascular Remodeling

Objective— Vascular remodeling is a physiological process that occurs in response to long-term changes in hemodynamic conditions, but may also contribute to the pathophysiology of intima-media thickening (IMT) and vascular disease. Shear stress detection by the endothelium is thought to be an important determinant of vascular remodeling. Previous work showed that platelet endothelial cell adhesion molecule-1 (PECAM-1) is a component of a mechanosensory complex that mediates endothelial cell (EC) responses to shear stress. Methods and Results— We tested the hypothesis that PECAM-1 contributes to vascular remodeling by analyzing the response to partial carotid artery ligation in PECAM-1 knockout mice and wild-type littermates. PECAM-1 deficiency resulted in impaired vascular remodeling and significantly reduced IMT in areas of low flow. Inward remodeling was associated with PECAM-1-dependent NFB activation, surface adhesion molecule expression, and leukocyte infiltration as well as Akt activation and vascular cell proliferation. Conclusions— PECAM-1 plays a crucial role in the activation of the NFB and Akt pathways and inflammatory cell accumulation during vascular remodeling and IMT. Elucidation of some of the signals that drive vascular remodeling represent pharmacologically tractable targets for the treatment of restenosis after balloon angioplasty or stent placement.

[Integrative Physiology/Experimental Medicine] Fibronectin Is an Important Regulator of Flow-Induced Vascular Remodeling

Objective— Fibronectin is an important regulator of cell migration, differentiation, growth, and survival. Our data show that fibronectin also plays an important role in regulating extracellular matrix (ECM) remodeling. Fibronectin circulates in the plasma and is also deposited into the ECM by a cell dependent process. To determine whether fibronectin affects vascular remodeling in vivo, we asked whether the fibronectin polymerization inhibitor, pUR4, inhibits intima-media thickening, and prevents excess ECM deposition in arteries using a mouse model of vascular remodeling. Methods and Results— To induce vascular remodeling, partial ligation of the left external and internal carotid arteries was performed in mice. pUR4 and the control peptide were applied periadventitially in pluronic gel immediately after surgery. Animals were euthanized 7 or 14 days after surgery. Morphometric analysis demonstrated that the pUR4 fibronectin inhibitor reduced carotid intima (63%), media (27%), and adventitial thickening (40%) compared to the control peptide (III-11C). Treatment with pUR4 also resulted in a dramatic decrease in leukocyte infiltration into the vessel wall (80%), decreased ICAM-1 and VCAM-1 levels, inhibited cell proliferation (60% to 70%), and reduced fibronectin and collagen I accumulation in the vessel wall. In addition, the fibronectin inhibitor prevented SMC phenotypic modulation, as evidenced by the maintenance of smooth muscle (SM) -actin and SM myosin heavy chain levels in medial cells. Conclusions— These data are the first to demonstrate that fibronectin plays an important role in regulating the vascular remodeling response. Collectively, these data suggest a therapeutic benefit of periadventitial pUR4 in reducing pathological vascular remodeling.

[Integrative Physiology/Experimental Medicine] Reduced Collagen Biosynthesis Is the Hallmark of Cerebral Aneurysm: Contribution of Interleukin-1{beta} and Nuclear Factor-{kappa}B

Background— Reduced extracellular matrix is a prominent feature of cerebral aneurysms (CAs). We previously reported excessive ECM degradation in CA walls. In the present study, we examined collagen biosynthesis in CA walls and the molecular mechanisms underlying it in CA progression. Methods and Results— RT-PCR and immunohistochemistry showed reduced expression of procollagen type I, III, and lysyl oxidase (LOX) in CA walls. Treatment with the LOX inhibitor β-aminopropionitrile resulted in enhanced progression of CA. Expression of procollagen type I, III, and LOX was inhibited by interleukin-1β (IL-1β) in cultured rat aortic smooth muscle cells (RASMCs) in vitro. Nuclear factor -B (NF-B) was activated in IL-1β-stimulated RASMCs, and treatment with NF-B decoy oligodeoxynucleotides (ODN) restored reduced expression of procollagen type I, III, and LOX in vitro. NF-B decoy ODNs ameliorated the expression of procollagen type I, III, and LOX in CA walls in vivo. Conclusions— Collagen biosynthesis was significantly inhibited at the transcriptional level and in the posttranscriptional enzymatic modification in CA walls through upregulated expression of IL-1β and the NF-B pathway. Reduced collagen biosynthesis may contribute to CA progression, and inhibition of this process may lead to the prevention of the progression and rupture of CAs.

[Integrative Physiology/Experimental Medicine] Activated Protein C Protects Against Myocardial Ischemia/ Reperfusion Injury via Inhibition of Apoptosis and Inflammation

Objective— In spite of major advances in reperfusion therapy for patients presenting with acute coronary syndrome, long-term morbidity is still substantial. A limitation of initial treatment of myocardial ischemia is the lack of prevention of ischemia/reperfusion (I/R) injury. Activated protein C (APC), a crucial mediator in the coagulation process, plays a prominent role in the crosstalk between coagulation and inflammation and provides cytoprotective effects via inhibition of apoptosis and inflammation in several human and animal studies. Methods and Results— APC was administered in an animal model for myocardial I/R. APC largely inhibited early myocardial I/R injury after varying reperfusion times, an effect that was absent on administration of heparin, a nonspecific anticoagulant agent. The protective effects of APC were absent in case of absence or blockade of protease activated receptor-1 (PAR-1), indicating a critical role for PAR-1 in this process. Furthermore, we showed a strong antiapoptotic effect of APC in the early phase of reperfusion combined with an antiinflammatory effect at an early stage (IL-6), as well as at a later stage (leukocyte infiltration). Conclusions— APC exerts strong protective effects on early myocardial I/R injury, primarily via inhibition of apoptosis and inflammation, which are regulated via PAR-1.

[Integrative Physiology/Experimental Medicine] Preconditioning by Mitochondrial Reactive Oxygen Species Improves the Proangiogenic Potential of Adipose-Derived Cells-Based Therapy

Objective— Transplantation of adipose-derived stroma cells (ADSCs) stimulates neovascularization after experimental ischemic injury. ADSC proangiogenic potential is likely mediated by their ability to differentiate into endothelial cells and produce a wide array of angiogenic and antiapoptotic factors. Mitochondrial reactive oxygen species (ROS) have been shown to control ADSC differentiation. We therefore hypothesized that mitochondrial ROS production may change the ADSC proangiogenic properties. Methods and Results— The use of pharmacological strategies (mitochondrial inhibitors, antimycin, and rotenone, with or without antioxidants) allowed us to specifically and precisely modulate mitochondrial ROS generation in ADSCs. We showed that transient stimulation of mitochondrial ROS generation in ADSCs before their injection in ischemic hindlimb strongly improved revascularization and the number of ADSC-derived CD31-positive cells in ischemic area. Mitochondrial ROS generation increased the secretion of the proangiogenic and antiapoptotic factors, VEGF and HGF, but did not affect ADSC ability to differentiate into endothelial cells, in vitro. Moreover, mitochondrial ROS-induced ADSC preconditioning greatly protect ADSCs against oxidative stress-induced cell death. Conclusion— Our study demonstrates that in vitro preconditioning by moderate mitochondrial ROS generation strongly increases in vivo ADSC proangiogenic properties and emphasizes the crucial role of mitochondrial ROS in ADSC fate.

[Cell Biology/Signaling] Induction and Isolation of Vascular Cells From Human Induced Pluripotent Stem Cells--Brief Report

Objective— Induced pluripotent stem (iPS) cells are a novel stem cell population derived from human adult somatic cells through reprogramming using a defined set of transcription factors. Our aim was to determine the features of the directed differentiation of human iPS cells into vascular endothelial cells (ECs) and mural cells (MCs), and to compare that process with human embryonic stem (hES) cells. Methods and Results— We previously established a system for differentiating hES cells into vascular cells. We applied this system to human iPS cells and examined their directed differentiation. After differentiation, TRA1–60– Flk1+ cells emerged and divided into VE-cadherin-positive and -negative populations. The former were also positive for CD34, CD31, and eNOS and were consistent with ECs. The latter differentiated into MCs, which expressed smooth muscle -actin and calponin after further differentiation. The efficiency of the differentiation was comparable to that of human ES cells. Conclusions— We succeeded in inducing and isolating human vascular cells from iPS cells and indicate that the properties of human iPS cell differentiation into vascular cells are nearly identical to those of hES cells. This work will contribute to our understanding of human vascular differentiation/development and to the development of vascular regenerative medicine.

[Cell Biology/Signaling] Notch Signaling Induces Osteogenic Differentiation and Mineralization of Vascular Smooth Muscle Cells: Role of Msx2 Gene Induction via Notch-RBP-Jk Signaling

Objective— Vascular calcification is closely correlated with cardiovascular morbidity and mortality. Here, we demonstrate the role of Notch signaling in osteogenic differentiation and mineralization of vascular smooth muscle cells (SMCs). Methods and Results— The Msx2 gene, a key regulator of osteogenesis, was highly induced by coculture with Notch ligand-expressing cells or overexpression of Notch intracellular domains (NICDs) in human aortic SMCs (HASMCs). Furthermore, the Notch1 intracellular domain (N1-ICD) overexpression markedly upregulated alkaline phosphatase (ALP) activity and matrix mineralization of HASMCs. A knockdown experiment with a small interfering RNA confirmed that Msx2 mediated N1-ICD–induced osteogenic conversion of HASMCs. Interestingly, Msx2 induction by N1-ICD was independent of bone morphogenetic protein–2 (BMP-2), an osteogenic morphogen upstream of Msx2. The transcriptional activity of the Msx2 promoter was significantly enhanced by N1-ICD overexpression. The RBP-Jk binding element within the Msx2 promoter was critical to Notch-induced Msx2 gene expression. Correspondingly, N1-ICD overexpression did not induce the Msx2 expression in RBP-Jk–deficient fibroblasts. Immunohistochemistry of human carotid artery specimens revealed localization of Notch1, Jagged1 and Msx2 to fibrocalcific atherosclerotic plaques. Conclusion— These results imply a new mechanism for osteogenic differentiation of vascular SMCs in which Notch/RBP-Jk signaling directly induces Msx2 gene expression and suggest its crucial role in mediating vascular calcification.

[Cell Biology/Signaling] Sonic Hedgehog Induces Notch Target Gene Expression in Vascular Smooth Muscle Cells via VEGF-A

Objective— Notch, VEGF, and components of the Hedgehog (Hh) signaling pathway have been implicated in vascular morphogenesis. The role of Notch in mediating hedgehog control of adult vascular smooth muscle cell (SMC) growth and survival remains unexplored. Methods and Results— In cultured SMCs, activation of Hh signaling with recombinant rShh (3.5 µg/mL) or plasmid encoded Shh increased Ptc1 expression, enhanced SMC growth and survival and promoted Hairy-related transcription factor (Hrt) expression while concomitantly increasing VEGF-A levels. These effects were significantly reversed after Hh inhibition with cyclopamine. Shh-induced stimulation of Hrt-3 mRNA and SMC growth and survival was attenuated after inhibition of Notch-mediated CBF-1/RBP-Jk-dependent signaling with RPMS-1 while siRNA knockdown of Hrt-3 inhibited SMC growth and survival. Recombinant VEGF-A increased Hrt-3 mRNA levels while siRNA knockdown abolished rShh stimulated VEGF-A expression while concomitantly inhibiting Shh-induced increases in Hrt-3 mRNA levels, proliferating cell nuclear antigen (PCNA), and Notch 1 IC expression, respectively. Hedgehog components were expressed within intimal SMCs of murine carotid arteries after vascular injury concomitant with a significant increase in mRNA for Ptc1, Gli2, VEGF-A, Notch 1, and Hrts. Conclusion— Hedgehog promotes a coordinate regulation of Notch target genes in adult SMCs via VEGF-A.

[Cell Biology/Signaling] Rab GTPase Regulation of VEGFR2 Trafficking and Signaling in Endothelial Cells

Objective— Vascular endothelial growth factor receptor 2 (VEGFR2) is a receptor tyrosine kinase that regulates vascular physiology. However, mechanism(s) by which VEGFR2 signaling and trafficking is coordinated are not clear. Here, we have tested endocytic Rab GTPases for regulation of VEGFR2 trafficking and signaling linked to endothelial cell migration. Methods and Results— Quiescent VEGFR2 displays endosomal localization and colocalization with the Rab5a GTPase, an early endosome fusion regulator. Expression of GTP or GDP-bound Rab5a mutants block activated VEGFR2 trafficking and degradation. Manipulation of Rab7a GTPase activity associated with late endosomes using overexpression of wild-type or mutant proteins blocks activated VEGFR2 trafficking and degradation. Depletion of Rab7a decreased VEGFR2 Y1175 phosphorylation but increased p42/44 (pERK1/2) MAPK phosphorylation. Endothelial cell migration was increased by Rab5a depletion but decreased by Rab7a depletion. Conclusions— Rab5a and Rab7a regulate VEGFR2 trafficking toward early and late endosomes. Our data suggest that VEGFR2-mediated regulation of endothelial function is dependent on different but specific Rab-mediated GTP hydrolysis activity required for endosomal trafficking.

[Cell Biology/Signaling] Stimulation of Cell Surface F1-ATPase Activity by Apolipoprotein A-I Inhibits Endothelial Cell Apoptosis and Promotes Proliferation

Objectives— Several findings argue for a protective effect of high-density lipoproteins (HDLs) against endothelial dysfunction. The molecular mechanisms underlying this protective effect are not fully understood, although recent works suggest that the actions of HDL on the endothelium are initiated by multiple interactions between HDLs (lipid or protein moiety) and cell surface receptors. We previously showed that the mitochondrial related F1-ATPase is a cell surface receptor for HDLs and their main atheroprotective apolipoprotein (apoA-I). Herein we test the hypothesis that the cell surface F1-ATPase may contribute to the ability of apoA-I and HDLs to maintain endothelial cell survival. Methods and Results— Cell imaging and binding assays confirmed the presence of the F1-ATPase at the surface of human umbilical vein endothelial cells (HUVECs) and its ability to bind apoA-I. Cell surface F1-ATPase activity (ATP hydrolysis into ADP) was stimulated by apoA-I and was inhibited by its specific inhibitor IF1-H49K. Furthermore the antiapoptotic and proliferative effects of apoA-I on HUVECs were totally blocked by the F1-ATPase ligands IF1-H49K, angiostatin and anti-βF1-ATPase antibody, independently of the scavenger receptor SR-BI and ABCA1. Conclusion— This study suggests an important contribution of cell surface F1-ATPase to apoA-I-mediated endothelial cell survival, which may contribute to the atheroprotective functions of apoA-I.

[Cell Biology/Signaling] COX-2 Limits Prostanoid Production in Activated HUVECs and Is a Source of PGH2 for Transcellular Metabolism to PGE2 by Tumor Cells

Objective— Inducible expression of cyclooxygenase-2 (COX-2) and terminal prostaglandin synthases (tPGS) has been mainly analyzed in tumor, stromal, and inflammatory cells, and little is known about the regulation of prostanoid biosynthesis by endothelial cells. Here we characterize the profile of prostanoids produced by activated HUVECs and analyze the expression and activities of tPGS. Methods and Results— Enzyme immunoassays indicated increased endothelial prostanoid production after proangiogenic stimulation, but without parallel upregulation of tPGS. Endothelial prostanoid production instead depended on the induction of COX-2 and was abolished by COX-2 silencing or pharmacological inhibition. COX-2 is functionally coupled to prostacyclin and thromboxane synthases in HUVECs, but these cells show no detectable PGE2 synthase (PGES) activity. Endothelial PGE2 production is partly mediated by nonenzymatic decomposition of COX-2-derived PGH2, but endothelial-produced PGH2 can also be metabolized enzymatically by microsomal PGES-1 in cocultured tumor cells. Conclusions— Our findings identify a novel transcellular metabolism of PGE2 between the endothelial and tumor compartments. Given the role of PGE2 as a mediator of COX-2 proangiogenic effects, transcellular metabolism of endothelial-derived PGH2 is a potential target for treatment of pathological angiogenesis.

[Cell Biology/Signaling] Augmentation of Megakaryocyte Expression of Fc{gamma}RIIa by Interferon {gamma}

Objective— The purpose of this study was to identify factors that alter expression of FcRIIa by megakaryocytes. Methods and Results— Effects of selected cytokines and growth factors on megakaryocyte expression of FcRIIa were assessed with phorbol 12-myristate 13-acetate (PMA)-differentiated human erythroleukemia (HEL) cells and with thrombopoietin-differentiated CD34 stem cells and compared with those obtained with myelocytic cell lines and a monocytic cell lines. Expression of FcRIIa was quantified with the use of Western blots and real-time reverse transcriptase-polymerase chain reaction. Megakaryocyte differentiation was identified by expression of CD41, CD42, and von Willebrand factor with the use of flow cytometry. Interferon (IFN) increased protein expression of FcRIIa by HEL cells and CD34 stem cells after megakaryocyte differentiation (maximal 4-fold, P<0.001 for each). IFN did not increase expression of FcRIIa by undifferentiated HEL and CD34 cells. Expression of FcRIIa mRNA was increased (2-fold, P<0.001) in megakaryocyte-differentiated HEL cells. IFN did not increase expression of FcRIIa by undifferentiated myelocytic or monocytic cell lines. Conclusions— IFN appears to selectively increase expression of FcRIIa by cells exhibiting characteristics of megakaryocytes. This effect of IFN may contribute to greater platelet expression of FcRIIa in patients with atherosclerotic vascular disease.

[Clinical and Population Studies] Serum Resistin Concentrations and Risk of New Onset Heart Failure in Older Persons: The Health, Aging, and Body Composition (Health ABC) Study

Objective— Resistin is associated with inflammation and insulin resistance and exerts direct effects on myocardial cells including hypertrophy and altered contraction. We investigated the association of serum resistin concentrations with risk for incident heart failure (HF) in humans. Methods and Results— We studied 2902 older persons without prevalent HF (age, 73.6±2.9 years; 48.1% men; 58.8% white) enrolled in the Health, Aging, and Body Composition (Health ABC) Study. Correlation between baseline serum resistin concentrations (20.3±10.0 ng/mL) and clinical variables, biochemistry panel, markers of inflammation and insulin resistance, adipocytokines, and measures of adiposity was weak (all rho <0.25). During a median follow-up of 9.4 years, 341 participants (11.8%) developed HF. Resistin was strongly associated with risk for incident HF in Cox proportional hazards models controlling for clinical variables, biomarkers, and measures of adiposity (HR, 1.15 per 10.0 ng/mL in adjusted model; 95% CI, 1.05 to 1.27; P=0.003). Results were comparable across sex, race, diabetes mellitus, and prevalent and incident coronary heart disease subgroups. In participants with available left ventricular ejection fraction at HF diagnosis (265 of 341; 77.7%), association of resistin with HF risk was comparable for cases with reduced versus preserved ejection fraction. Conclusions— Serum resistin concentrations are independently associated with risk for incident HF in older persons.