bluuebunny/crossref_metadata_2025 huggingface dataset:
Fondaparinux and enoxaparin in comparison to unfractionated heparin in preventing thrombus formation on mechanical heart valves in an ex vivo rabbit model
Authors: Buerke Michael , Hauroeder Baerbel , Peetz Dirk , Hundt Ferdinand , Bickel Christoph , Schaefer Iris , Meyer Juergen , Rupprecht Hans J. , Schlitt Axel
Published: July 2003

The aim of the present study was to investigate the efficacy of three different parenterally administered anticoagulants for the prevention of thrombus formation on artificial heart valves in an experimental rabbit model.

Unfractionated heparin was administered intravenously in group I (n = 10), Enoxaparin subcutaneously in group II (n = 10), fondaparinux intravenously in group III (n = 10), and no medication was administered to group IV (n = 9). Leaflets from Sulzer Carbomedics bileaflet mechanical heart valves were placed in a flow chamber. The flow chamber was filled with blood in a continuous circulation between the carotid artery and the jugular vein.

In group IV the flow chamber was clotted after a median of 15 minutes of circulation. Weight analysis before and after 1 h of perfusion showed that the median thrombus weight was 18.0 mg in group I, 17.7 mg in group II, 20.3 mg in group III, and 30.8 mg in group IV. Further analysis by electron microscopy showed similar results regarding deposition of fibrin, platelets, and erythrocytes on leaflet surfaces.

Fondaparinux and subcutaneously administered enoxaparin were as effective as intravenously administered unfractionated heparin in preventing thrombus formation on artificial heart valve leaflets in our investigation. This rabbit model, in which the heart valve leaflets were exposed to rabbit blood for a short time under laminar flow, should be further evaluated with respect to whether it can provide information about anti-thrombotic regimens in patients after mechanical heart valve replacement.
Biochemical importance of glycosylation of plasminogen activator inhibitor-1
Authors: Gils Ann , Pedersen Katrine E. , Skottrup Peter , Christensen Anni , Naessens Dominik , Deinum Johanna , Enghild Jan J. , Declerck Paul J. , Andreasen Peter A.
Published: July 2003

The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous glycosylation pattern of the sites at N209 and N265, while that at N329 is not utilised. The IC50-values for inactivation of PAI-1 by 4 monoclonal antibodies differed strongly between glycosylated PAI-1 and non-glycosy-lated PAI-1 expressed in E. coli. For 3 antibodies, an overlap of the epitopes with the glycosylation sites could be excluded as explanation for the differential reactivity. The latency transition of non-glycosylated, but not of glycosylated PAI-1, was strongly accelerated by a non-ionic detergent. The different biochemical properties of glycosylated and non-glycosylated PAI-1 depended specifically on glycosylation of either one or the other of the utilised sites. The PAI-1-binding protein vitronectin reversed the changes associated with the lack of glycosylation at one of the sites. Our results stress the importance of the source of PAI-1 when studying the mechanisms of action of PAI-1-inactivating compounds of potential clinical importance.
Affinity and kinetics of P-selectin binding to heparin
Authors: Wang Jian-Guo , Geng Jian-Guo
Published: July 2003

P-selectin (CD62P), expressed on stimulated endothelial cells and activated platelets, reacts with P-selectin glycoprotein ligand-1 (PSGL-1, CD162) for leukocyte rolling. It also binds to heparin and heparan sulfate proteoglycans (HSPGs), which attenuates P-selectin mediated adhesions of leukocytes and cancer cells. Here we report that P-selectin mediated adhesion, but not rolling, of the HSPGs bearing human malignant melanoma A375 cells under shear stress. To understand its underlying molecular mechanism, we measured the biophysical properties of this interaction. Heparin inhibited the adhesion of A375 cells to immobilized P-selectin under flow (IC50 = 3 µM heparin) and neutralized the binding of P-selectin to A375 cells (IC50 = 4 µM heparin). Using surface plasmon resonance technique, we found that P-selectin bound to heparin with a dissociation constant (Kd
) of 115 ± 6 nM. The measured off rate (k
off) was 3.15 ± 0.34 × 10-3 s-1 and the calculated on rate (k
on) was 2.75 × 104 M-1 s-1. Taken together, our data suggest that the very slow k
off and the reduced k
on, but apparently not the Kd
, are responsible for adhesion, but not rolling of A375 cells, to P-selectin under flow.
Type II transmembrane serine proteases
Authors: Szabo Roman , Wu Qingyu , Dickson Robert B. , Netzel-Arnett Sarah , Antalis Toni M. , Bugge Thomas H.
Published: July 2003

The recent availability of human and mouse genome sequences and expressed sequence tag databases facilitated the identification of a large new family of membrane anchored serine proteases, the type II transmembrane serine proteases or TTSPs. Analyses of human inherited disorders and gene targeting studies in mice have revealed that several members of this new protease family have critical functions in development and health. Preliminary studies also suggest that aberrant expression of type II transmembrane serine proteases may be linked to disease progression. The knowledge gathered thus far of the genetics, physiology, and pathology of this interesting new serine protease family will be reviewed here in brief.
VEGF-induced HUVEC migration and proliferation are decreased by PDE2 and PDE4 inhibitors
Authors: Favot Laure , Keravis Thérèse , Holl Vincent , Bec Alain Le , Lugnier Claire
Published: July 2003

Migration and proliferation of endothelial cells in response to VEGF play an important role in angiogenesis associated to pathologies such as atherosclerosis, diabetes and tumor development. Elevation of cAMP in endothelial cells has been shown to inhibit growth factor-induced proliferation. Our hypothesis was that inactivation of cAMP-specific phosphodiesterases (PDEs) would inhibit angiogenesis. The purpose of this study was to evaluate the effect of PDE inhibitors on in vitro and in vivo angiogenesis, using human umbilical vein endothelial cell (HUVEC) and chick chorioallantoic membrane (CAM) models respectively. Here, we report that: 1) PDE2, PDE3, PDE4 and PDE5 are expressed in HUVEC; 2) EHNA (20 µM), PDE2 selective inhibitor, and RP73401 (10 µM), PDE4 selective inhibitor, are able to increase the intracellular cAMP level in HUVEC; 3) EHNA and RP73401 are able to inhibit proliferation, cell cycle progression and migration of HUVEC stimulated by VEGF; 4) these in vitro effects can be mimic by treating HUVEC with the cAMP analogue, 8-Br-cAMP (600 µM); 5) only the association of EHNA and RP73401 inhibits in vivo angiogenesis, indicating that both migration and proliferation must be inhibited. These data strongly suggest that PDE2 and PDE4 represent new potential therapeutic targets in pathological angiogenesis.
Inhibition of thrombosis in melanoma allografts in mice by endogenous mast cell heparin
Authors: Corwin Mark , Yu Hon , Wang Jun , Nalcioglu Orhan , Su Min-Ying , Samoszuk Michael
Published: July 2003

An unexplained paradox of malignant melanoma is the apparent failure of the blood within the tumor to clot despite the presence of multiple factors that should promote blood clotting. Here we present histochemical evidence that human and murine melanomas are extensively infiltrated by abundant mast cells. Because mast cells contain the natural anticoagulant heparin, the present studies were aimed at defining the role of mast cell heparin in preventing the blood from clotting within B16 melanoma grafts in C57BL/6 J mice. Mice bearing B16 melanoma grafts were treated with non-specific or specific inhibitors of mast cell heparin (protamine or heparinase, respectively). After the drug treatment there was histologic and functional evidence of selective thrombosis of the blood vessels within the protamine and heparinase treated melanoma grafts. A similar, high degree of thrombosis was also observed in B16 tumors grown in transgenic NDST-2 knockout mice bearing a targeted disruption in the gene coding for mast cell heparin synthesis. The tumors grown in the protamine-treated animals were significantly smaller than the tumors from control (untreated mice). By contrast, the tumors treated with heparinase or grown in the NDST-2 knockout mice were significantly larger than the tumors from control (untreated) mice. We conclude that the intrinsic procoagulant properties of malignant melanoma are neutralized in vivo by the anticoagulant properties of endogenous heparin produced by mast cells that naturally infiltrate the tumor. Our results also suggest that thrombosis and hemostasis within melanoma may play a complex role in modulating the growth of the tumor.
The role of PAR4 in thrombin-induced thromboxane production in human platelets
Authors: Wu Chin-Chung , Hwang Tsong-Long , Liao Chang-Hui , Kuo Sheng-Chu , Lee Fang-Yu , Teng Che-Ming
Published: July 2003

There are two protease-activated receptors (PARs), PAR1 and PAR4, in human platelets. It has been suggested that PAR1 mediates platelet responses to low concentrations of thrombin, whereas PAR4 mediates signaling only at high concentrations. In the present study, we used a selective PAR4 blocker, YD-3, to investigate the role of PAR4 in thrombin-induced thromboxane formation in human platelets. YD-3 completely prevented thromboxane production by either a low concentration of thrombin (0.1 U/ml) or the PAR4 agonist peptide GYPGKF. In contrast, YD-3 did not affect thromboxane production caused by the PAR1 agonist peptide SFLLRN, collagen or arachidonic acid. YD-3 also decreased [3H]arachidonic acid release from thrombin-stimulated platelets. Moreover, desensitization of platelets with GYPGKF prevented low thrombin-induced thromboxane formation. The decreased thromboxane production by YD-3 is linked to inhibition of calcium influx in thrombin-stimulated platelets. These results suggest that PAR4 plays an important role in the regulation of thromboxane formation in platelets responding to thrombin through prolonged elevation of [Ca2+]i and activation of phospholipase A2. These data also indicate that PAR4 can be activated by relatively low concentrations of thrombin in human platelets. The selective inhibition of thrombin-induced thromboxane production by YD-3 may be of therapeutic benefit for thrombotic diseases.
Enhanced TNFα and oxidative stress in patients with heart failure: effect of TNFα on platelet O2
- production
Authors: Biase Luciano De , Pignatelli Pasquale , Lenti Luisa , Tocci Giuliano , Piccioni Fabiana , Riondino Silvia , Pulcinelli Fabio M. , Rubattu Speranza , Volpe Massimo , Violi Francesco
Published: July 2003

Experimental studies have suggested that TNFα, a pro-inflammatory cytokine, may contribute to the deterioration of cardiovascular function through various mechanisms, including the generation of reactive oxygen species. It has not yet been demonstrated whether TNFα has prooxidant activity in patients with heart failure, and what the mechanism eventually resulting in this effect are.

We analyzed 42 patients (38 men and 4 women, aged 26 to 74 years) with heart failure, secondary to idiopathic dilated car-diomyopathy (n=21), coronary artery disease (n=15), and valve disease (n=6), and 20 controls (18 men and 2 women, aged 49 to 67 years). Ten patients were in class I,9 in class II,15 in class III and 8 in class IV according to NYHA Classification. Blood samples were obtained from each patient to evaluate basal and collagen-induced platelet O2
- production, and plasma TNFα.

In vivo results showed increased platelet O2
- production and plasma TNFα levels in NYHA class III-IV compared with that in controls or in NYHA I-II (p<0,001); platelet O2
- production correlated significantly (R=0,6; p<0,01) with TNFα plasma levels. In vitro studies showed TNFα dose-dependently (5-40 pg/ml) induced platelet O2
- production, and that this effect was significantly inhibited by its specific inhibitor, WP9QY (1 μM); aspirin (100 μM), AACOCF3, a specific PLA2 inhibitor (14 μM), and DPI, an inhibitor of NADPH oxidase, significantly inhibited TNFα-mediated platelet O2
- production.

This study suggests that in patients with heart failure, enhanced platelet O2
- production is mediated by TNFα via activation of arachidonic acid and NADPH oxidase pathways.
Prevention of thrombosis following deep arterial injury in rats by bovine activated protein C requiring co-administration of bovine protein S
Authors: Dahlbäck Björn , Arnljots Björn , Malm Karl
Published: July 2003

The antithrombotic effect of bovine activated protein C (bAPC) given with or without bovine protein S (bPS) was investigated in a rat model of deep arterial injury. A segment of the left common carotid artery was isolated between vascular clamps and opened longitudinally. An endarterectomy was performed and the arteriotomy was closed with a running suture, whereafter the vessel was reperfused by removing the clamps. The antithrombotic effect (vascular patency rates 31 minutes after reperfusion) and the arteriotomy bleeding were measured. Ten treatment groups each containing 10 rats and a control group of 20 animals were in a blind random fashion given intravenous bolus injections of increasing doses of activated protein C, with or without co-administration of protein S. The groups received either bAPC alone (0.8, 0.4, 0.2 or 0.1 mg/kg), bAPC (0.8, 0.4, 0.2, 0.1 or 0.05 mg/kg) combined with bPS (0.6 mg/kg), or bPS alone (0.6 mg/kg) whereas the control group received vehicle only. Administered alone, bAPC or bPS had no antithrombotic effect, regardless of dosage. In contrast, all groups that were treated with bAPC in combination with bPS demonstrated a significant antithrombotic effect, as compared to controls. Neither bAPC, bPS, nor the combination of bAPC and bPS increased the arteriotomy bleeding significantly compared to controls. In vitro clotting assays using bAPC or bPS alone yielded only minor prolongation of clotting time, whereas bAPC combined with bPS prolonged the clotting time considerably, demonstrating the dependence on the APC-cofactor activity of bPS for expression of anticoagulant activity by bAPC. In conclusion, our study shows the in vivo significance of protein S as a cofactor to activated protein C, and that potent anti-thrombotic effect can be achieved by low doses of bAPC combined with bPS, without producing hemorrhagic side effects.
Lymphangiogenic growth factors, receptors and therapies
Authors: Lohela Marja , Saaristo Anne , Veikkola Tanja , Alitalo Kari
Published: July 2003

The lymphatic vasculature is essential for the maintenance of normal fluid balance and for the immune responses, but it is also involved in a variety of diseases. Hypoplasia or dysfuction of the lymphatic vessels can lead to lymphedema, whereas hyperplasia or abnormal growth of these vessels are associated with lymphangiomas and lymphangiosarcomas. Lymphatic vessels are also involved in lymph node and systemic metastasis of cancer cells. Recent novel findings on the molecular mechanisms involved in lymphatic vessel development and regulation allow the modulation of the lymphangiogenic process and specific targeting of the lymphatic endothelium.

Recent results show that the homeodomain transcription factor Prox-1 is an important lymphatic endothelial cell (LEC) fate-determining factor which can induce LEC-specific gene transcription even in blood vascular endothelial cells (BECs). This suggests that the distinct phenotypes of cells in the adult vascular endothelium are plastic and sensitive to transcriptional reprogramming, which might be useful for future therapeutic applications involving endothelial cells

Vascular endothelial growth factor-C (VEGF-C) and VEGF-D are peptide growth factors capable of inducing the growth of new lymphatic vessels in vivo in a process called lymphangiogenesis. They belong to the larger family which also includes VEGF, placenta growth factor (PlGF) and VEGF-B. VEGF-C and VEGF-D are ligands for the endothelial cell specific tyrosine kinase receptors VEGFR-2 and VEGFR-3. In adult human as well as mouse tissues VEGFR-3 is expressed predominantly in lymphatic endothelial cells which line the inner surface of lymphatic vessels. While VEGFR-2 is thought to be the main mediator of angiogenesis, VEGFR-3 signaling is crucial for the development of the lymphatic vessels. Heterozygous inactivation of the VEGFR-3 tyrosine kinase leads to primary lymphedema due to defective lymphatic drainage in the limbs. Other factors that seem to be involved in lymphangiogenesis include the Tie/angiopoietin system, neuropilin-2 and integrin α9.

VEGF-C induces lymphatic vessel growth, but high levels of VEGF-C also resulted in blood vessel leakiness and growth. The VEGFR-3-specific mutant form of VEGF-C called VEGF-C156S lacks blood vascular side effects but is sufficient for therapeutic lymphangiogenesis in a mouse model of lymphedema. As VEGF-C156S is a specific lymphatic endothelial growth factor in the skin, it provides an attractive molecule for pro-lymphangiogenic therapy.

This publication was partially financed by Serono. Part of this paper was originally presented at the 2nd International Workshop on New Therapeutic Targets in Vascular Biology, which took place in Geneva, Switzerland from February 6-9, 2003.
Temporal Relationships Between Circulating Levels of CC and CXC Chemokines and Developing Atherosclerosis in Apolipoprotein E*3 Leiden Mice
Authors: Murphy Nuala , Bruckdorfer K. Richard , Grimsditch David C. , Overend Philip , Vidgeon-Hart Martin , Groot Pieter H.E. , Benson G. Martin , Graham Annette
Published: July 2003

Objectives—

CC and CXC chemokines are implicated in leukocyte recruitment during development of atherosclerotic lesions, suggesting circulating levels of chemokines may be useful serum markers of atherogenesis. Serum chemokine concentrations were measured in apolipoprotein (apo) E*3 Leiden mice and their nontransgenic littermates and related to the differing rates of atherogenesis in these animals.

Methods and Results—

Mice were fed a high-fat, high-cholesterol/cholate (HFC/C) diet for 18 weeks. Circulating levels of JE/monocyte chemotactic protein-1 increased (
P
<0.05) after 2 to 4 weeks, coincident with development of diet-induced hypercholesterolemia, and remained elevated throughout the study. Circulating KC concentrations increased (
P
<0.05) after consumption of HFC/C diet; however, unlike JE, serum KC concentrations increased more rapidly in apoE*3 Leiden mice than their nontransgenic littermates. Hepatic expression of JE and KC mRNA were detected by in situ hybridization in all mice fed HFC/C diet. Aortic expression of JE mRNA was seen only in apoE*3 Leiden mice within macrophage-rich atherosclerotic lesions. By contrast, no aortic expression of KC mRNA was detected by in situ hybridization.

Conclusions—

Increases in serum chemokine concentrations did not reflect temporal aortic production of these molecules and proved less predictive than serum cholesterol of the markedly different extent of atheroma in apoE*3 Leiden and nontransgenic mice.
Regulation of the Expression of the Apolipoprotein(a) Gene
Authors: Huby Thierry , Afzal Veena , Doucet Chantal , Lawn Richard M. , Gong Elaine L. , Chapman M. John , Thillet Joëlle , Rubin Edward M.
Published: July 2003

Objective—

The apolipoprotein(a) [apo(a)] gene locus is the major determinant of the circulating concentration of the atherothrombogenic lipoprotein Lp(a). In vitro analysis of the intergenic region between the apo(a) and plasminogen genes revealed the presence of a putative apo(a) transcription control region (ACR) approximately 20 kb upstream of the apo(a) gene that significantly increases the minimal promoter activity of the human apo(a) gene.

Methods and Results—

To examine the function of the ACR in its natural genomic context, we used the Cre-
lox
P recombination system to generate 2 nearly identical apo(a)–yeast artificial chromosome transgenic mouse lines that possess a single integration site for the human apo(a) transgene in the mouse genome but differ by the presence or absence of the ACR enhancer. Analysis of the 2 groups of animals revealed that the deletion of the ACR was associated with 30% reduction in plasma and mRNA apo(a) levels. Apo(a)–yeast artificial chromosome transgenic mice with and without the ACR sequence were similar in all other aspects of apo(a) regulation, including liver-specific apo(a) expression and alteration in expression levels in response to sexual maturation and a high-fat diet.

Conclusions—

This study provides the first experimental in vivo evidence for a functional role of the ACR enhancer in determining levels of apo(a) expression.
Intimal Retention of Cholesterol Derived From Apolipoprotein B100– and Apolipoprotein B48–Containing Lipoproteins in Carotid Arteries of Watanabe Heritable Hyperlipidemic Rabbits
Authors: Proctor Spencer D. , Mamo John C.L.
Published: July 2003

Objectives—

The arterial retention of apolipoprotein (apo) B100- and apoB48-containing lipoproteins was simultaneously determined in a rabbit model of human hypercholesterolemia using 3D confocal microscopy.

Methods and Results—

Lipoproteins containing apoB100 (LDL) and apoB48 (chylomicron remnants) were differentially conjugated with fluorophores and simultaneously perfused at equivalent concentrations under physiological conditions in situ through carotid vessels of Watanabe heritable hyperlipidemic rabbits and compared with controls. Retention of lipoproteins was defined as the amount remaining after an extensive washout phase. LDL and chylomicron remnants were both retained, primarily within the subendothelial space. Without a concomitant increase in exposure to lipoproteins, we found a marked increase in the retention of cholesterol within the intima of Watanabe heritable hyperlipidemic rabbits compared with controls, specifically because of increased entrapment of apoB48-containing lipoproteins.

Conclusions—

Collectively, our data suggest that hypercholesterolemia induced as a consequence of LDL receptor deficiency differentially influences retention of LDL and of chylomicron remnants.
TR3 Orphan Receptor Is Expressed in Vascular Endothelial Cells and Mediates Cell Cycle Arrest
Authors: Arkenbout E. Karin , van Bragt Maaike , Eldering Eric , van Bree Chris , Grimbergen Jos M. , Quax Paul H.A. , Pannekoek Hans , de Vries Carlie J.M.
Published: July 2003

Objective—

Endothelial cells play a pivotal role in vascular homeostasis. In this study, we investigated the function of the nerve growth factor–induced protein-B (NGFI-B) subfamily of nuclear receptors comprising the TR3 orphan receptor (TR3), mitogen-induced nuclear orphan receptor (MINOR), and nuclear orphan receptor of T cells (NOT) in endothelial cells.

Methods and Results—

The mRNA expression of TR3, MINOR, and NOT in atherosclerotic lesions was assessed in human vascular specimens. Each of these factors is expressed in smooth muscle cells, as described before, and in subsets of endothelial cells, implicating that they might affect endothelial cell function. Adenoviral overexpression of TR3 in cultured endothelial cells resulted in decreased [
3
H]thymidine incorporation, whereas a dominant-negative TR3 variant that inhibits the activity of endogenous TR3-like factors enhanced DNA synthesis. TR3 interfered with progression of the cell cycle by upregulating p27
Kip1
and downregulating cyclin A, whereas expression levels of a number of other cell cycle–associated proteins remained unchanged.

Conclusions—

These findings demonstrate that TR3 is a modulator of endothelial cell proliferation and arrests endothelial cells in the G1 phase of the cell cycle by influencing cell cycle protein levels. We hypothesize involvement of TR3 in the maintenance of integrity of the vascular endothelium.
Human CRP Gene Polymorphism Influences CRP Levels
Authors: Brull D.J. , Serrano Norma , Zito F. , Jones Lisa , Montgomery H.E. , Rumley A. , Sharma Pankaj , Lowe G.D.O. , World M.J. , Humphries S.E. , Hingorani A.D.
Published: July 2003

Objective—

C-reactive protein (CRP) concentrations are predictive of cardiovascular disease, and levels are heritable, in part. We identified novel polymorphisms in the CRP gene and assessed their influence on CRP level.

Methods and Results—

CRP was measured in 250 male army recruits before and after strenuous exercise and perioperatively in 193 coronary artery bypass graft (CABG) patients. Two novel polymorphisms were identified in the CRP gene, −717G>A in the promoter and +1444C>T in the 3′UTR. Among army recruits, CRP was higher in +1444TT homozygotes than +1444 C-allele carriers at baseline (1.04±0.38 versus 0.55±0.06,
P
=0.014) and at all time points after exercise (2.35±0.68 versus 1.07±0.12, 2.11±0.53 versus 0.88±0.09, and 1.77±0.44 versus 0.71±0.09,
P
=0.034,
P
=0.007, and
P
=0.013, at 2, 48, and 96 hours after exercise, respectively). In the CABG patients, mean CRP (mg/L) rose from 1.97±0.36 at baseline to 167.2±5.0 72 hours postoperatively. Genotype did not influence CRP at baseline; however, peak post-CABG CRP levels were higher in +1444TT homozygotes compared with +1444C-allele carriers (198±17 versus 164±5,
P
=0.03).

Conclusions—

The CRP gene +1444C>T variant influences basal and stimulated CRP level. These findings have implications both for the prediction and pathogenesis of coronary heart disease.
Chlamydia pneumoniaeBinds to Platelets and Triggers P-Selectin Expression and Aggregation
Authors: Kälvegren Hanna , Majeed Meytham , Bengtsson Torbjörn
Published: July 2003

Objective—Evidence linkingChlamydia pneumoniaeto atherosclerotic cardiovascular disease is expanding. Platelets are considered to play an essential role in cardiovascular diseases; however, so far platelets have not been associated with an infectious cause of atherosclerosis. This study aims to clarify the interaction betweenC pneumoniaeand platelets and possibly present a novel mechanism in the pathogenesis of atherosclerosis.

Methods and Results—The effects ofC pneumoniaeon platelet aggregation and secretion were assessed with lumiaggregometry, and the ability ofC pneumoniaeto bind to platelets and stimulate expression of P-selectin was analyzed with flow cytometry. We found thatC pneumoniae, at a chlamydia:platelet ratio of 1:15, adheres to platelets and triggers P-selectin expression after 1 minute and causes an extensive aggregation and ATP secretion after 20 minutes of incubation. Inhibition of glycoprotein IIb/IIIa with Arg-Gly-Asp-Ser or abciximab markedly reducedC pneumoniae-induced platelet aggregation. Exposure ofC pneumoniaeto polymyxin B, but not elevated temperature, abolished the stimulatory effects on platelet activation, suggesting that chlamydial lipopolysaccharide has an active role. In contrast, other tested bacteria had no or only moderate effects on platelet functions.

Conclusion—Our findings demonstrate a new concept of howC pneumoniaeactivates platelets and thereby may cause atherosclerosis and thrombotic vascular occlusion.
Coronary Plaque Morphology and Frequency of Ulceration Distant From Culprit Lesions in Patients With Unstable and Stable Presentation
Authors: Schoenhagen Paul , Stone Gregg W. , Nissen Steven E. , Grines Cindy L. , Griffin John , Clemson Barry S. , Vince D. Geoffrey , Ziada Khaled , Crowe Timothy , Apperson-Hanson Carolyn , Kapadia Samir R. , Tuzcu E. Murat
Published: July 2003

Objective—

Intravascular ultrasound studies describe ruptured coronary plaques at sites remote from the culprit lesion in patients with acute myocardial infarction (MI), suggesting multifocal plaque vulnerability. However, the role of intravascular ultrasound in the diagnosis of lesion vulnerability before rupture is unclear.

Methods and Results—

We compared morphology and frequency of ulceration of additional plaques proximal to the culprit lesion in 105 patients treated with emergent stenting during an evolving, acute MI in the CADILLAC study and 92 patients with stable/subacute presentation who underwent elective stenting. Additional plaques proximal to the culprit lesion were found in 52 (50%) and 54 (59%) patients in the acute MI and stable/subacute group, respectively. The prevalence of ulceration was significantly higher in the acute MI than in the stable/subacute group (19% versus 4%;
P
=0.014). However, there was no significant difference in other morphological lesion characteristics.

Conclusions—

Additional plaques are frequently found adjacent to the culprit lesions in patients undergoing percutaneous coronary intervention independent of clinical presentation. The increased prevalence of plaque ulceration but otherwise similar morphology of additional lesions in patients with acute MI versus stable/subacute presentation demonstrates the limitations of imaging in the assessment of plaque vulnerability.
Macrophage-Specific p53 Expression Plays a Crucial Role in Atherosclerosis Development and Plaque Remodeling
Authors: Merched Aksam J. , Williams Elizabeth , Chan Lawrence
Published: July 2003

Objective—

We first showed that absence of p53 accelerates atherosclerosis development in apoE-deficient mice. In this study, we investigated how macrophage-specific loss of p53 function might modulate atherosclerosis development in LDL receptor-deficient mice, a model for familial hypercholesterolemia.

Methods and Results—

We transferred bone marrow cells isolated from p53
+/+
and p53
−/−
mice to lethally irradiated LDL receptor
−/−
mice and evaluated the aortic atherosclerotic lesion areas in the recipients at different times afterward. At 15 weeks and again at 20 weeks, we found larger aortic lesion size in mice receiving p53
−/−
cells compared with those that received p53
+/+
cells. By measuring the rate of bromodeoxyuridine incorporation, we found that the absence of p53 in macrophages stimulates cellular proliferation. In contrast, the rate of apoptosis in the atheromatous lesion was similar in the two groups of mice. Furthermore, we found that the absence of macrophage-specific p53 expression was associated with vulnerable-appearing lesions marked by increased tissue necrosis and reduced collagen deposition.

Conclusions—

p53 plays a crucial role in atherosclerosis lesion development and remodeling, and macrophage-specific p53 deficiency stimulates cellular proliferation leading to a vulnerable-appearing phenotype of lesions in a mouse model of familial hypercholesterolemia.
Atrial Natriuretic Peptide Regulates Regional Vascular Volume and Venous Tone in Humans
Authors: Schmitt Matthias , Broadley Andrew J.M. , Nightingale Angus K. , Payne Nicola , Gunaruwan Prasad , Taylor Justin , Lee Leong , Cockcroft John , Struthers Allan D. , Frenneaux Michael P.
Published: July 2003

Objective—

To date, the contribution of basal atrial natriuretic peptide (ANP) levels to resting vascular function in humans is unknown. In the present study we sought to investigate the role of ANP in regulating regional vascular volume and venous tone in healthy subjects.

Methods and Results—

We used radionuclide plethysmography to examine the effects of ANP and the ANP-receptor antagonist A71915 on forearm vascular volume. Creating pressure/volume relations, we determined changes in vascular volume, compliance, and tone. Performing dose-ranging studies, we additionally assessed the potency and specificity of A71915 in the forearm resistance vasculature. Equilibrium blood pool scintigraphy was then used to assess the effects of systemic administration of A71915 on regional intestinal vascular volume. Infusion of ANP increased forearm vascular volume in a dose-dependent manner (maximum 20%;
P
<0.001), exerting a maximum venodilating effect at plasma levels similar to that seen in heart failure. A71915 increased venous tone, thereby decreasing vascular volume by 9.6±1.1%,
P
<0.001 (forearm), and 2.6±0.5%,
P
=0.01 (intestinal beds). At an infusion ratio of 50:1, A71915 almost completely abolished the effects of ANP on forearm blood flow.

Conclusions—

ANP locally regulates regional vascular volume and tone without affecting compliance.
Emerging Anticoagulant Drugs
Authors: Bates Shannon M. , Weitz Jeffrey I.
Published: July 2003

Abstract—The limitations of traditional anticoagulants, heparin and warfarin, have prompted the development of new anticoagulant drugs for prevention and treatment of both venous and arterial thromboembolism. After a brief review of thrombogenesis and its regulation, this study focuses on new anticoagulant agents in more advanced stages of clinical testing.
Grb2 Is Required for the Development of Neointima in Response to Vascular Injury
Authors: Zhang Shaosong , Ren Jie , Khan M. Faisal , Cheng Alec M. , Abendschein Dana , Muslin Anthony J.
Published: July 2003

Objective—

Neointima formation occurs in arteries in response to mechanical or chemical injury and is responsible for substantial morbidity. In this work, the role of the intracellular linker protein Grb2 in the pathogenesis of neointima formation was examined. Grb2 is a critical signaling protein that facilitates the activation of the small GTPase ras by receptor tyrosine kinases.

Methods and Results—

Cultured rat aortic smooth muscle cells were treated with an antisense morpholino to Grb2 and these cells showed a reduced proliferative response to platelet-derived growth factor stimulation.
Grb2
−/−
mice do not survive embryonic development.

Grb2
+/−

mice appear normal at birth and are fertile but have defective signaling in several tissues. Cultured smooth muscle cells derived from
Grb2
+/−
mice grew at a much slower rate than cells derived from
Grb2
+/+
mice.
Grb2
+/−
and
Grb2
+/+
mice were subjected to carotid injury. After 21 days,
Grb2
+/+
mice developed robust neointima formation that, in some cases, resulted in an occlusive lesion. In contrast,
Grb2
+/−
mice were resistant to the development of neointima

Conclusions—

Grb2 is an essential component of the signaling cascade resulting in neointima formation after arterial injury.
Soluble Elastin Fragments in Serum Are Elevated in Acute Aortic Dissection
Authors: Shinohara Tadashi , Suzuki Kimihiro , Okada Makoto , Shiigai Masaru , Shimizu Masashi , Maehara Tadaaki , Ohsuzu Fumitaka
Published: July 2003

Objective—

We aimed to establish an enzyme-linked immunosorbent assay for measuring soluble elastin fragments (sELAF) in serum and to reveal its usefulness in diagnosing acute aortic dissection (AAD).

Methods and Results—

An enzyme-linked immunosorbent assay to measure sELAF in serum was developed by using the newly created double monoclonal antibodies, which recognize the different epitopes of human aortic elastin. Twenty-five AAD patients, 50 patients with acute myocardial infarction (AMI), and 474 healthy individuals were enrolled in the study. The sELAF levels from healthy subjects gradually increased with aging. When the cutoff point for positivity was set at the mean+3 SD (ie, 3 SD above the mean in healthy subjects at each age), 16 AAD patients (64.0%) were found be positive, whereas only 1 AMI patient was found to be positive (2.0%). AAD patients with either an open or a partially open pseudolumen were found be 88.9% positive for sELAF, whereas those with its early closure were 0% positive. The difference in the sELAF levels between AAD patients with and without a thrombotic closure of false lumen was significant (60.3±15.6 versus 135.4±53.2 ng/mL, respectively;
P
<0.005).

Conclusions—

The sELAF level in serum may be a useful marker for helping in the diagnosis and screening of AAD and may also help to distinguish AAD from AMI.
VLDL Induces Adipocyte Differentiation in ApoE-Dependent Manner
Authors: Chiba Tsuyoshi , Nakazawa Toru , Yui Katsumasa , Kaneko Eiji , Shimokado Kentaro
Published: July 2003

Objective—To clarify the role of very low density lipoprotein (VLDL) and apolipoprotein E (apoE) in adipogenesis, we studied newly developed hyperlipidemic obese (ob/ob;apoE−/−) mice. Because hydrolysis of VLDL is believed to be the major source of adipogenic free fatty acids, a higher plasma level of VLDL in these mice should exaggerate obesity.

Methods and Results—When fed a high-fat, high-cholesterol diet,ob/ob;apoE−/−mice did not show increased body weight or an increased amount of adipose tissue in spite of increased plasma VLDL levels, whereasob/obmice showed an increased body weight and amount of adipose tissue, suggesting that there is a novel apoE-dependent pathway for adipogenesis. In vitro experiments using bone marrow stromal cells and 3T3-L1 cells confirmed this notion. ApoE-deficient VLDL did not induce adipogenesis, whereas normal VLDL induced adipogenesis in these cells. The incubation of apoE-deficient VLDL with recombinant human apoE restored its adipogenic activity. Tetrahydrolipstatin, a lipoprotein lipase inhibitor, did not affect the adipogenic activity of VLDL, suggesting that hydrolysis of VLDL did not play a major role in its effects. In fact, lipid components of VLDL or free fatty acids induced only partial adipogenesis.

Conclusions—Our findings indicate that VLDL induces adipogenesis in an apoE-dependent manner both in vitro and in vivo.
Predictive Value of D-Dimer Test for Recurrent Venous Thromboembolism After Anticoagulation Withdrawal in Subjects With a Previous Idiopathic Event and in Carriers of Congenital Thrombophilia
Authors: Palareti Gualtiero , Legnani Cristina , Cosmi Benilde , Valdré Lelia , Lunghi Barbara , Bernardi Francesco , Coccheri Sergio
Published: July 2003

Background—

We have shown that normal D-dimer levels obtained after the discontinuation of oral anticoagulant treatment (OAT) has a high negative predictive value for recurrent venous thromboembolism (VTE). The aim of the present study was to assess the predictive value of D-dimer for recurrent VTE in subjects with a previous unprovoked event who are either carriers of inherited thrombophilia or not.

Methods and Results—

We prospectively evaluated 599 patients (301 males) with a previous VTE episode. They were repeatedly examined for D-dimer levels after OAT withdrawal and were screened for inherited thrombophilic alterations. Alterations were detected in 130 patients (21.7%), factor V Leiden (70 patients; 2 of whom were homozygotes) and prothrombin mutation (38 patients) were the most prevalent ones. Recurrent events were recorded in 58 subjects (9.7%) during a follow-up of 870.7 patient-years. Altered D-dimer levels at 1 month after OAT withdrawal were associated with a higher rate of subsequent recurrence in all subjects investigated, especially in those with an unprovoked qualifying VTE event (hazard ratio, 2.43; 95% confidence interval, 1.18 to 4.61) and in those with thrombophilia (hazard ratio, 8.34; 95% confidence interval, 2.72 to 17.43). The higher relative risk for recurrence of altered D-dimer was confirmed by multivariate analysis after adjustment for other risk factors. The negative predictive value of D-dimer was 92.9% and 95.8% in subjects with an unprovoked qualifying event or with thrombophilia, respectively.

Conclusions—

D-dimer levels measured 1 month after OAT withdrawal have a high negative predictive value for recurrence in subjects with unprovoked VTE who are either carriers or not carriers of congenital thrombophilia.
N
-Terminal Pro–Brain Natriuretic Peptide and Other Risk Markers for the Separate Prediction of Mortality and Subsequent Myocardial Infarction in Patients With Unstable Coronary Artery Disease
Authors: James Stefan K. , Lindahl Bertil , Siegbahn Agneta , Stridsberg Mats , Venge Per , Armstrong Paul , Barnathan Elliot S. , Califf Robert , Topol Eric J. , Simoons Maarten L. , Wallentin Lars
Published: July 2003

Background—

Biochemical markers are useful for prediction of cardiac events in patients with non–ST-segment-elevation acute coronary syndrome (ACS). The associations between
N
-terminal pro–brain natriuretic peptide (NT-proBNP) and other biochemical and clinical risk indicators, as well as their prognostic value concerning the individual end points of death and myocardial infarction (MI), were elucidated in a large cohort of ACS patients.

Methods and Results—

NT-proBNP, troponin T, and C-reactive protein (CRP) were analyzed in blood samples obtained at a median of 9.5 hours from symptom onset in 6809 of 7800 ACS patients in the Global Utilization of Strategies To Open occluded arteries-IV (GUSTO-IV) trial. Levels of NT-proBNP were correlated independently with age, female gender, low body weight, diabetes, renal dysfunction, history of MI, heart failure, heart rate, ongoing myocardial damage, and time since onset of ischemia. Increasing quartiles of NT-proBNP were related to short- and long-term mortality that reached 1.8%, 3.9%, 7.7%, and 19.2%, (
P
<0.001), respectively, at 1 year. Levels of troponin T, CRP, heart rate, and creatinine clearance, in addition to ST-segment depression, were also correlated independently with 1-year mortality, but NT-proBNP was the marker with the strongest relation. In contrast, only troponin T, creatinine clearance, and ST-segment depression were independently related to future MI. The combination of NT-proBNP and creatinine clearance provided the best prediction, with a 1-year mortality of 25.7% with both markers in the top quartile vs 0.3% with both markers in the bottom quartile.

Conclusions—

The use of NT-proBNP appears to add critical prognostic insight to the assessment of patients with ACS.
β
1
-Adrenergic Receptor Blockade Attenuates Angiotensin II–Mediated Catecholamine Release Into the Cardiac Interstitium in Mitral Regurgitation
Authors: Tallaj José , Wei Chih-Chang , Hankes Gerald H. , Holland Merrilee , Rynders Patricia , Dillon A. Ray , Ardell Jeffrey L. , Armour J. Andrew , Lucchesi Pamela A. , Dell’Italia Louis J.
Published: July 2003

Background—

This study tested the hypothesis that β
1
-adrenoreceptor blockade modulates the angiotensin II (Ang II)–evoked neural release of norepinephrine (NE) and epinephrine (Epi) into the cardiac interstitial fluid (ISF) space in experimentally induced mitral regurgitation (MR) in the dog.

Methods and Results—

Normal dogs (n=8) were compared with dogs with MR of 2 (n=8) and 4 (n=6) weeks’ duration and with dogs with MR treated with β
1
-receptor blockade (RB; extended-release metoprolol succinate, 100 mg QD; MR+β
1
-RB) that was started 24 hours after MR induction for 2 (n=6) and 4 weeks (n=8). Left ventricular end-diastolic dimension increased 20% as plasma Ang II levels increased >5-fold in both MR and MR+β
1
-RB dogs at 2 and 4 weeks. Ang II infusion into the left atrium produced increases in ISF NE and Epi in normal dogs, which were further increased in 2- and 4-week MR dogs but were restored to normal in 4-week MR+β
1
-RB dogs. Ang II infusion produced 4-fold increases in circulating NE and Epi in 2- and 4-week MR dogs that returned to normal in 4-week+β
1
-RB dogs. Left ventricular angiotensin-converting enzyme activity and ISF Ang II were increased in 4-week MR dogs but were decreased in 4-week MR+β
1
-RB dogs.

Conclusions—

β
1
-RB decreases renin-angiotensin system sympathostimulation and activation by attenuating the Ang II–mediated NE and Epi release into the cardiac ISF and circulation and by decreasing left ventricular angiotensin-converting enzyme expression in the early phases of volume overload.
Inflammation Modifies the Effects of a Reduced-Fat Low-Cholesterol Diet on Lipids
Authors: Erlinger Thomas P. , Miller Edgar R. , Charleston Jeanne , Appel Lawrence J.
Published: July 2003

Background—

Inflammatory mediators regulate key aspects of lipid metabolism. We hypothesized that inflammation could diminish the cholesterol-lowering effect of a reduced-fat/low-cholesterol diet.

Methods and Results—

After a 2-week run-in period on a control diet (37% total fat, 16% saturated fat), 100 participants were randomized to the control or DASH diet (27% total fat, 6% saturated fat) for 12 weeks. Median C-reactive protein (CRP) at baseline was 2.37 mg/L (interquartile range, 1.20, 3.79). The DASH diet, net of control, had no effect on CRP. Overall, there were significant net reductions in total (−0.34 mmol/L), LDL (−0.29 mmol/L), and HDL (−0.12 mmol/L) cholesterol from the DASH diet (each,
P
<0.001) and little change in triglycerides (+0.05 mmol/L,
P
=0.21). Baseline CRP was strongly associated with lipid responsiveness to the DASH diet. Total and LDL cholesterol were reduced to a greater degree in those with a “low” (below median) compared with a “high” (above median) baseline CRP (total, −9.8% versus −3%;
P
for interaction=0.006; LDL cholesterol, −11.8% versus −3%;
P
for interaction=0.009). Reductions in HDL cholesterol (−8.8%) were similar in persons with low versus high CRP. Triglycerides were increased in those with a high CRP but not in those with a low CRP (19.8% versus +0%;
P
for interaction=0.019).

Conclusions—

In this study, the presence of increased CRP was associated with less total and LDL cholesterol reduction and a greater increase in triglycerides from a reduced-fat/low-cholesterol diet. These findings document an additional mechanism by which inflammation might increase cardiovascular disease risk.
Sphingosine Kinase Modulates Microvascular Tone and Myogenic Responses Through Activation of RhoA/Rho Kinase
Authors: Bolz Steffen-Sebastian , Vogel Lukas , Sollinger Daniel , Derwand Roland , Boer Christa , Pitson Stuart M. , Spiegel Sarah , Pohl Ulrich
Published: July 2003

Background—

RhoA and Rho kinase are important modulators of microvascular tone.

Methods and Results—

We tested whether sphingosine kinase (Sphk1) that generates the endogenous sphingolipid mediator sphingosine-1-phosphate (S1P) is part of a signaling cascade to activate the RhoA/Rho kinase pathway. Using a new transfection model, we report that resting tone and myogenic responses of isolated resistance arteries increased with forced expression of Sphk1 in smooth muscle cells of these arteries. Overexpression of a dominant negative Sphk1 mutant or coexpression of dominant negative mutants of RhoA or Rho kinase together with Sphk1 completely inhibited development of tone and myogenic responses.

Conclusions—

The tone-increasing effects of a Sphk1 overexpression suggest that Sphk1 may play an important role in the control of peripheral resistance.