bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic deletion of tissue-type plasminogen activator (t-PA) in APOE3-Leiden mice reduces progression of cholesterol-induced atherosclerosis
Authors: Gijbels Marion , Offerman Erik , Rezaee Farhad , Verheijen Jan
Published: September 2003

During recent years it has become increasingly recognized that the plasmin activation system is involved in the development of atherosclerosis.

In this paper, we have studied the contribution of the plasmino-gen activation system in the development of atherosclerosis by cross-breeding apoE3-Leiden mice, which have a human-like lipid profile, with mice deficient in PAI-1 (plasminogen-activator inhibitor-1), u-PA (urokinase plasminogen activator), and t-PA (tissue plasminogen activator). Genetic compound offspring was used to evaluate the progression of atherosclerotic lesions after they were fed a variant atherogenic diet for 12 weeks. Lesion area of plaques in the aortic valve was not significantly different in apoE3-Leiden:PAI -/- and apoE3-Leiden:u-PA -/-mice as compared to apoE3-Leiden mice. In contrast, a significant 70 % reduction of the lesion area was observed in apoE3-Leiden:t-PA -/- mice as compared to control group apoE3-Leiden mice. In addition the early, regular fatty streaks and mild plaques increased in apoE3-Leiden:t-PA -/- mice, whereas the severe plaques (type IV and V) decreased in these animals.

A lower deposition of collagen was observed in the atherosclerotic lesions of apoE3-Leiden:t-PA -/- mice as compared with apoE3-Leiden mice. Our results indicate for the first time that t-PA deficiency delayed the atherosclerotic process in this mouse model.
Does recombinant factor VIIa, apart from overall hemostasis, regulate TAFI dependent fibrinolysis? In vitro analysis using overall hemostasis potential (OHP) assay
Authors: Antovic Aleksandra , Antovic Jovan
Published: September 2003

Using the recently developed overall hemostatic potential (OHP) assay, we investigated the effects of different concentrations of recombinant factor VIIa on overall hemostasis and on thrombin activatable fibrinolysis inhibitor (TAFI) dependent fibrinolysis in different factor deficient plasmas. rFVIIa increased OHP in FV, FVIII and FIX deficient plasmas but not up to the levels in normal pooled plasma (NPP). Maximal levels were found at 2.4 µg/mL of rFVIIa, while higher doses did not further increase OHP. In FXII deficient plasma, OHP is higher than in NPP and addition of rVIIa further increased it. Even very high concentrations of rFVIIa (9.6 µg/mL) did not induce a significant increase of OHP in NPP. Higher concentrations of rVIIa down-regulated fibrinolysis in FVIII, FIX and FXI deficient plasmas to values obtained in NPP. Using potato tuber carboxy-peptidase inhibitor (PTCI), a specific inhibitor of TAFI, it was found that TAFI’s influence on fibrinolysis down-regulation in FVIII and FIX deficient plasmas increased after addition of higher concentrations of rFVIIa.

From this in vitro study it seems that rFVIIa in a concentration of 2.4 µg/mL improves overall hemostasis in FV, FVIII and FIX deficient plasmas. rFVIIa, even at very high (supra-therapeutic) concentrations, does not induce hypercoagulability either in NPP or in deficient plasmas. Higher concentrations of rVIIa induce, at least partly, TAFI dependent down-regulation of fibrinolysis in FVIII and FIX deficient plasmas. These results, together with some previous ex vivo studies, point to OHP assay as a possible diagnostic tool for evaluating the hemostatic effects of rFVIIa.
Evidence for functionally active protease-activated receptor-3 (PAR-3) in human vascular smooth muscle cells
Authors: Spanbroek Rainer , Lötzer Katharina , Richard Habenicht Andreas , Schrör Karsten , Bretschneider Ellen
Published: September 2003

The present study investigates whether vascular smooth muscle cells of the human saphenous vein (SMC) express a functionally active protease-activated receptor-3 (PAR-3). PAR-3 mRNA was detected by RT-PCR. In the presence of thrombin, a rapid and transient increase in PAR-3 mRNA was observed. Stimulation of SMC with thrombin or the synthetic PAR-3-activating peptide, TFRGAP, resulted in transient mobilization of intracellular calcium. After a preceding challenge with thrombin, the calcium signal to TFRGAP was abolished, suggesting cleavage and subsequent desensitization of PAR-3 by thrombin. Activation of PAR-3 by TFRGAP elicited a time-dependent activation of the extracellular-signal-regulated kinase (ERK)-1/2 with a maximum response 10-20 min after stimulation. At 200 µM, TFRGAP increased 3H]-thymidine incorporation into cellular DNA about two-fold. These data indicate that PAR-3 is expressed in human SMC and triggers intracellular signaling. Thus, in the SMC PAR-3 might contribute to thrombin-induced responses.
Sprouty proteins: antagonists of endothelial cell signaling and more
Authors: Cabrita Miguel , Christofori Gerhard
Published: September 2003

Among many signaling pathways, receptor tyrosine kinases (RTKs) can activate the mitogen-activated protein kinase (MAPK) signaling pathway that subsequently leads to a variety of cellular changes, including proliferation, differentiation and motility. The regulation of growth factor signaling is complex, and various cell types respond differently to the same stimulus for reasons not entirely understood. The recent discovery in Drosophila of Sprouty (dSpry), an inhibitor of RTK-induced MAPK activation, provides clues to how these signals are regulated. In mammals, four orthologues of dSpry, Spry1-4, have been described, and in this review we discuss their functional characteristics. Mammalian Sprys, like dSpry, are ligand-induced feedback inhibitors of a number of growth factor receptors. In endothelial cells, upon fibroblast growth factor (FGF) receptor and vascular endothelial growth factor (VEGF) receptor activation, Sprys translocate to the plasma membrane and inhibit cell growth and proliferation. However, in epidermal growth factor (EGF)-stimulated cells, Sprys can enhance MAPK activation. In addition, Sprys have many binding partners, including different effectors of the MAPK activation pathway. The intersection point where Sprys interfere in the MAPK pathway as well as their interactions with other proteins may partly explain the dual, yet opposing roles, on growth factor-induced MAPK activation. Moreover, Sprys require tyrosine phosphorylation to interact with their binding partners, a prerequisite for their dual function. Hence, Sprys add another layer of complexity to the regulation of RTK-mediated signal transduction that begins to explain the variation in cellular responses to growth factors.

This publication was partially financed by Serono Foundation for the Advancement of Medical Science.

Part of this paper was originally presented at the 2nd International Workshop on New Therapeutic Targets in Vascular Biology from February 6-9, 2003 in Geneva, Switzerland.
AMP-activated protein kinase (AMPK) regulates the insulin-induced activation of the nitric oxide synthase in human platelets
Authors: Schulz Christian , Fichtlscherer Birgit , Kemp Bruce , Fisslthaler Beate , Busse Rudi , Fleming Ingrid
Published: September 2003

Little is known about the signaling cascades that eventually regulate the activity of the endothelial nitric oxide synthase (eNOS) in platelets. Here, we investigated the effects of insulin on the phosphorylation and activation of eNOS in washed human platelets and in endothelial cells.

Insulin activated the protein kinase Akt in cultured endothelial cells and increased the phosphorylation of eNOS on Ser1177but failed to increase endothelial cyclic GMP levels or to elicit the relaxation of endothelium-intact porcine coronary arteries. In platelets, insulin also elicited the activation of Akt as well as the phosphorylation of eNOS and initiated NO production which was associated with increased cyclic GMP levels and the inhibition of thrombin-induced aggregation. The insulin-induced inhibition of aggregation was accompanied by a decreased Ca2+response to thrombin and was also prevented by Nωnitro-L- arginine. In platelets, but not in endothelial cells, insulin induced the activation of the AMP-activated protein kinase (AMPK), a metabolic stress-sensing kinase which was sensitive to the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin and the AMPK inhibitor iodotubercidin. Moreover, the insulin-mediated inhibition of thrombin-induced aggregation was prevented by iodotubercidin. Insulin-independent activation of the AMPK using 5-aminoimidazole-4-carboxamide ribonucleoside, increased platelet eNOS phosphorylation, increased cyclic GMP levels and attenuated platelet aggregation.

These results highlight the differences in the signal transduction cascade activated by insulin in endothelial cells and platelets, and demonstrate that insulin stimulates the formation of NO in human platelets, in the absence of an increase in Ca2+, by activating PI3-K and AMPK which phosphorylates eNOS on Ser1177.
Regulation of protease-activated receptor 1 (PAR1) on platelets and responsiveness to thrombin receptor activating peptide (TRAP) during systemic inflammation in humans
Authors: Reiter Rosemarie , Derhaschnig Ulla , Spiel Alexander , Keen Priska , Cardona Francesco , Mayr Florian , Jilma Bernd
Published: September 2003

Thrombin is a coagulation protease that activates platelets, endothelial cells, leukocytes and mesenchymal cells. Thrombin signaling is mediated at least in part by protease-activated receptors (PARs). As little is known about the in vivoregulation of PAR1, this study aimed to characterize the effects of systemic thrombin formation during human endotoxemia on the regulation of PAR1 and the associated responsiveness of human platelets to thrombin receptor activating peptide (TRAP). Endotoxin (2 ng/kg) was infused into 40 healthy men to study the regulation of PAR1 in systemic human inflammation. The SPAN12 antibody was used to determine the in vivoregulation of PAR1. To measure whether modulation of the PAR1 receptor may be associated with altered platelet reactivity, whole blood was stimulated with TRAP ex vivo. Thrombin generation was determined by prothrombin (F1+2) fragment. F1+2levels increased almost 9-fold from 0.5±0.1 nmol/L to 4.5±1.9 nmol/L at 4 h (p<0.001). PAR1 decreased by ~8% (p<0.001) within 2 h after endotoxin infusion and stayed at those levels until 6 h. Concomitantly, TRAP induced P-selectin expression maximally decreased by 18% (p<0.001) at 6 h. In conclusion, PAR1 expression is down-regulated on platelets during systemic thrombin formation induced by inflammation in humans which results in decreased responsiveness to subsequent stimulation of the PAR1 receptor.
Circulating endothelial cells in pulmonary hypertension
Authors: Golpon Heiko , Hebbel Robert , Solovey Anna , Cool Carlyne , Tuder Rubin , Geraci Mark , Voelkel Norbert , Bull Todd
Published: September 2003

The pulmonary endothelium plays a significant role in the pathobiology of Primary Pulmonary Hypertension. A number of diseases, related by a history of vascular injury, are associated with increased numbers of circulating endothelial cells (CECs). We hypothesized that patients with pulmonary hypertension would also have an increased number of circulating endothelial cells due to the high pressures and increased shear stress present within the pulmonary vasculature. We isolated the CECs from 14 patients with pulmonary hypertension, (5 primary and 11 secondary) and compared them to the cells from 12 normal controls. There was a significant increase in the number of CECs in peripheral blood in patients with both PPH and secondary pulmonary hypertension (SPH) when compared to normal volunteers (33.1 +/- 1.9 {PPH} and 27.2 +/- 6.9 {SPH} vs. 3.5 +/- 1.3 {controls}, p < 0.001). The number of circulating endothelial cells in the patient’s peripheral blood correlated significantly with the systolic, diastolic and mean pulmonary artery pressures of the individual. Approximately 50% of the CECs from patients with pulmonary hypertension expressed CD36, a marker of microvascular origin and 25% expressed E-selectin, a marker of endothelial cell activation. Although the origin of the CECs in patients with PH requires further investigation, one possible source is the pulmonary vasculature, and in patients with plexogenic pulmonary hypertension, the plexiform lesions. CECs may provide a non-invasive mean of accessing cells important to the pathobiology of severe pulmonary hypertension.
Plasmin generation dependent on α-enolase-type plasminogen receptor is required for myogenesis
Authors: López-Alemany Roser , Suelves Mònica , Muñoz-Cánoves Pura
Published: September 2003

Plasmin is a potent extracellular protease specialized in the degradation of fibrin (fibrinolysis). Active plasmin is generated by proteolytic activation of the zymogen plasminogen (Plg) by urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). α-Enolase constitutes a receptor for plasminogen on several leukocyte cell types, serving to localize and promote plasminogen activation pericellularly. However, a role for α-enolase-type plasminogen receptor (PlgR) in myogenesis has never been demonstrated. In this study, we show that C2C12 mouse myoblasts express PlgR, being its expression greatly induced during the differentiation process. A monoclonal antibody against PIgR MAb 11G1, with cell surface-generated plasmin inhibitory abilities, was able to fully abrogate C2C12 myoblast fusion and differentiation in vitro. Moreover, both plasmin activity and PlgR expression were significantly induced in regenerating skeletal muscle in vivo, either in experimentally-injured muscle or in the dystrophic muscle of mdx mouse (an animal model of human Duchenne muscular dystrophy, DMD). The mdx muscle presents better regeneration capacities and less fibrosis than the human DMD muscle; therefore, the increase in PlgR/plasmin activity in mdx muscle suggests an important contribution of the fibrinolytic system in mdx regeneration. This study constitutes the first indication of α-enolase-type plasminogen receptor as an important component of skeletal myogenesis, by concentrating and enhancing plasmin generation on the cell surface.
Membrane-associated CD40L and sCD40L in atherothrombotic disease
Authors: X.Anand Sunil , Viles-Gonzalez Juan , Badimon Juan , Cavusoglu Erdal , Marmur Jonathan
Published: September 2003

Recent data suggests that CD40L is involved in the pathogenesis of atherothrombotic disease. This review will focus on the history of CD40L, its role in platelet-mediated pathogenesis of atherothrombotic disease, its association to clinical syndromes and procedures, and its role in determining plaque rupture.
Stejnulxin, a novel snake C-type lectin-like protein from Trimeresurus stejnegeri venom is a potent platelet agonist acting specifically via GPVI
Authors: Lee Wen-Hui , Du Xiao-Yan , Lu Qiu-Min , Clemetson Kenneth , Zhang Yun
Published: September 2003

Stejnulxin, a novel snake C-type lectin-like protein with potent platelet activating activity, was purified and characterized from Trimeresurus stejnegeri venom. Under non-reducing conditions, it migrated on a SDS-polyacrylamide gel with an apparent molecular mass of 120 kDa. On reduction, it separated into three polypeptide subunits with apparent molecular masses of 16 kDa (α), 20 kDa (β1) and 22 kDa (β2), respectively. The complete amino acid sequences of its subunits were deduced from cloned cDNAs. The N-terminal sequencing and cDNA cloning indicated that β1 and β2 subunits of stejnulxin have identical amino acid sequences and each contains two N-glycosylation sites. Accordingly, the molecular mass difference between β1 and β2 is caused by glycosylation heterogenity. The subunit amino acid sequences of stejnulxin are similar to those of convulxin, with sequence identities of 52.6% and 66.4% for the α and β, respectively. Stejnulxin induced human platelet aggregation in a dose-dependent manner. Antibodies against αIIbβ3 inhibited the aggregation response to stejnulxin, indicating that activation of αIIbβ3 and binding of fibrinogen are involved in stejnulxin-induced platelet aggregation. Antibodies against GPIbα or α2β1 as well as echicetin or rhodocetin had no significant effect on stejnulxin-induced platelet aggregation. However, platelet activation induced by stejnulxin was blocked by anti-GPVI antibodies. In addition, stejnulxin induced a tyrosine phosphorylation profile in platelets that resembled that produced by convulxin. Biotinylated stejnulxin bound specifically to platelet membrane GPVI.
Risk factors for venous thrombosis in the black population
Authors: Patel Raj , Ford Elizabeth , Thumpston Jill , Arya Roopen
Published: September 2003

Risk factors for venous thrombo-embolism (VTE) in the black population are poorly characterized. Of 142 black cases tested a genetic cause was identified in only 9.1%: 4.2% had protein C deficiency, 2.8% protein S deficiency, 0.7% antithrombin deficiency and 1.4% were heterozygous for FV Leiden. We hypothesised that elevated factor VIII levels constitute a candidate risk factor for venous thrombosis in the black population. Factor VIII (FVIII:C) levels were determined in 100 black patients with VTE and 100 black controls in a case-control study. Of the patients 34% had a FVIII:C above 228 IU/dL (the 90thcentile value in normal blacks) compared to 10% controls. Relative to those with FVIII:C below this value, odds ratio (OR) for risk of VTE was 4.64 (95% CI 2.02-10.85). When FVIII:C below 150 IU/dL was used as a comparator, OR was 11.1 (95% CI 4.29-29.43). There was evidence for a dose-response relationship. We propose that raised FVIII:C is a major risk factor for VTE in black subjects with prevalence and odds ratio exceeding those reported for white subjects.
Role of blood coagulation factor XIII in patients with acute pulmonary embolism. Correlation of factor XIII antigen levels with pulmonary occlusion rate, fibrinogen, D-dimer, and clot firmness
Authors: Kucher Nils , Schroeder Verena , Kohler Hans
Published: September 2003

In patients with acute pulmonary embolism (PE), pulmonary occlusion rate is directly related to D-dimer and inversely related to fibrinogen levels. The role of coagulation factor XIII (FXIII) levels in acute venous thromboembolism is not known. A total of 120 consecutive patients with suspected PE and VIDAS D-dimer levels >500 μg/L were investigated by helical computed tomography (CT). Pulmonary occlusion rate was assessed by CT using the modified Miller index. D-dimer, fibrinogen, and FXIII A- and B-subunit antigen levels were taken on admission. Thrombelastography (TEG) was performed in a subset of patients (n=12).

The 71 patients with PE had lower FXIII A-subunit levels than the 49 patients with excluded PE (78.6±24.5% vs. 91.3±28.8%, p=0.01). In both groups, FXIII A-subunit was inversely related to D-dimer levels. FXIII A-subunit correlated with fibrinogen levels in patients with PE but not in patients without PE. FXIII A-subunit decreased with increasing pulmonary occlusion rate. The risk of PE was increased in the presence of A-subunit levels < 60% (OR 7.0 [95% CI 1.4-35.3], p=0.019). Clot firmness determined by TEG was lower in patients with PE than in patients without PE.

In patients with PE, circulating FXIII A-subunit is decreased compared to patients with suspected but excluded PE. The higher the clot burden within the pulmonary arteries the lower the FXIII antigen. In these patients, direct relation of FXIII A-subunit to fibrinogen levels argues for significant consumption of these coagulation factors in PE. This consumption of FXIII can also be detected by a global coagulation test like TEG.
Increased plasma levels of plasminogen activator inhibitor-1 and soluble vascular cell adhesion molecule after triacylglycerol infusion in man
Authors: Krebs Michael , Geiger Margarethe , Polak Kaija , Vales Anja , Schmetterer Leopold , Wagner Oswald , Waldhäusl Werner , Roden Michael , Binder Bernd
Published: September 2003

Increased plasma plasminogen activator inhibitor-1 (PAI-1) has been implicated in the development of vascular disease. In type 2 diabetes mellitus high PAI-1 levels are associated with increased plasma concentrations of free fatty acids (FFA) and triacylglycerol indicating an association or a causal relationship. To answer that question, the effect of FFA/triacylglycerol on plasma PAI-1 was examined. Ten healthy male volunteers were studied for 6 h during infusion of triacylglycerol [1.5 ml/min]/heparin [0.2 IU/(kg·min)] (LIP; n=10), saline only (SAL; n=10), and saline/heparin (HEP; n=5). Plasma insulin concentrations were kept constant at ~35 pmol/l by intravenous soma-tostatin-insulin infusions and there was no significant change in plasma glucose levels during any of the study protocols. LIP increased plasma triacylglycerol and FFA ~3- (p<0.001) and ~8-(p<0.000001) fold, respectively, within 90 min. Baseline plasma PAI-1 measured by a bio-immunoassay was similar in HEP (11.4±2.8 ng/ml), SAL (16.6±3.6 ng/ml), and LIP studies (15.2±3.4 ng/ml). Since studies were initiated in the morning, PAI-1 decreased (p<0.025) over time following its normal diurnal variation to 6.4±2.0 ng/ml and 4.0±2.4 ng/ml at 360 min in SAL and HEP, respectively. During LIP, however, PAI-1 increased to ~2.6 fold higher levels than during SAL at 360 min (16.4±4.0 ng/ml, p<0.01). While tissue plasminogen activator (tPA) and adipsin, an adipocyte derived protease, were unaffected by LIP, changes in soluble vascular cell adhesion molecule-1 (sVCAM-1) were significantly correlated (p=0.02) with those seen for PAI-1. This suggests that hyperlipidemia independent of insulin and plasma glucose levels stimulates vascular tissue and in turn might induce an increase in plasma PAI-1. PAI-1 then could contribute to the development of atherothrombotic vascular disease.
DNA Methylation, Smooth Muscle Cells, and Atherogenesis
Authors: Hiltunen Mikko O. , Ylä-Herttuala Seppo
Published: September 2003

DNA methylation is a form of epigenetic modification of the genome that can regulate gene expression. Hypermethylation of CpG islands in the promoter areas leads to decreased gene expression, whereas promoters of actively transcribed genes remain nonmethylated. Because of cellular proliferation and monoclonality of at least some of the lesion cells, atherosclerotic lesions have been compared with benign vascular tumors.

1,2

However, although genetic and epigenetic background favors neoplastic transformation, atherosclerotic plaques never develop to malignant tumors. Among cancer cells, common features are genome-wide hypomethylation, which correlates with transformation and tumor progression. Recent studies have shown that DNA methylation changes occur also during atherogenesis and may contribute to the lesion development.
Mapping of ATP, Glucose, Glycogen, and Lactate Concentrations Within the Arterial Wall
Authors: Levin M. , Leppänen O. , Evaldsson M. , Wiklund O. , Bondjers G. , Björnheden T.
Published: September 2003

Objective—

In large- and medium-sized arteries, the diffusion distances for oxygen and nutrients are long. This has been suggested to make these vessels prone to develop local energy metabolic deficiencies that could contribute to atherogenesis. To validate this hypothesis, we introduced a new method to measure energy metabolites within the arterial wall at high spatial resolution.

Methods and Results—

Bioluminescence imaging was used to quantify local metabolite concentrations in cryosections of snap frozen (in vivo) and incubated pig carotid artery rings. Incubation at hypoxia resulted in increased lactate concentrations, whereas ATP, glucose, and glycogen concentrations were decreased, especially in the mid media, where concentrations of these metabolites were close to zero. In snap frozen arteries, glycogen concentrations were markedly higher in deep layers of the media than toward the lumen. ATP, glucose, and lactate were more homogenously distributed.

Conclusions—

Bioluminescence imaging is a new and powerful tool to assess arterial wall energy metabolism at high spatial resolution. Our experiments demonstrate heterogeneous distributions of energy metabolites under hypoxic in vitro conditions. Furthermore, we show that glycogen concentrations are higher in deep medial layers in vivo. This might represent a local adaptation to a low-oxygen microenvironment.
Induction of Connective Tissue Growth Factor by Angiotensin II
Authors: Iwanciw Dominika , Rehm Margot , Porst Markus , Goppelt-Struebe Margarete
Published: September 2003

Objective—

Angiotensin II is recognized as one of the major mediators of cardiovascular pathology. Because connective tissue growth factor (CTGF) is involved in the pathophysiologic processes underlying fibrotic diseases, its regulation by angiotensin II was investigated.

Methods and Results—

In the 2-kidney, 1-clip model of renovascular hypertension, increased expression of CTGF was detectable in the hypertrophic left ventricle. By activation of angiotensin II type 1 receptors, angiotensin II caused rapid expression of CTGF mRNA and protein in a human fibroblast cell line. Activation of the p42/44 mitogen-activated protein (MAP) kinase signaling pathway proved to be essential for angiotensin II–stimulated CTGF expression. Inhibition of MAP kinase activation by forskolin prevented CTGF induction. Inhibition of the isoprenylation of small GTPases by simvastatin or pretreatment of the cells with toxin B reduced basal CTGF expression below detection limits and prevented induction by angiotensin II. Specific interference with RhoA signaling by Y27632 primarily reduced basal CTGF expression. There was no significant reduction of expression of angiotensin II type 1 receptors by simvastatin. These data indicate cooperation between the Rho signaling and the angiotensin II–activated MAP kinase pathways.

Conclusions—

Direct induction of CTGF by angiotensin II is indicative of a role for CTGF in angiotensin II–mediated fibrosis and might be a target of antifibrotic interventions.
Interleukin-13 Upregulates Vasodilatory 15-Lipoxygenase Eicosanoids in Rabbit Aorta
Authors: Tang Xin , Spitzbarth Nancy , Kuhn Hartmut , Chaitidis Pavlos , Campbell William B.
Published: September 2003

Objective—

Vasorelaxation of rabbit aorta is mediated by factors released from the vascular endothelium. In the aortic endothelium, arachidonic acid (AA) is metabolized via the 15-lipoxygenase pathway to the vasodilatory compounds 11,12,15-trihydroxyeicosatrienoic acid (THETA) and 15-hydroxy-11,12-epoxyeicosatrienoic acid (HEETA). Interleukin-13 (IL-13) increases 15-lipoxygenase expression and activity in several types of cells. We tested the hypothesis that IL-13 upregulates the 15-lipoxygenase pathway in rabbit aorta by inducing 15-lipoxygenase expression, thus increasing vascular relaxation mediated by THETA and HEETA.

Methods and Results—

Aorta rings and cultured endothelial cells were treated with IL-13, and 15-lipoxygenase expression was analyzed by reverse transcription–polymerase chain reaction and immunoblotting. 15-Lipoxygenase expression was increased by IL-13 in a concentration- and time-dependent manner. Aortic rings were incubated with [
14
C]AA, and the metabolites were extracted and resolved by high-performance liquid chromatography. IL-13 treatment increased the production of 15-hydroxyeicosatetraenoic acid, HEETA, and THETA. Indomethacin-resistant vasorelaxation to AA was significantly greater in IL-13–treated vessels than in controls. The relaxation responses to sodium nitroprusside were not altered by IL-13 treatment.

Conclusions—

These data indicate that in the vascular endothelium, IL-13 induces the expression of 15-lipoxygenase and increases the production of the vasodilatory eicosanoids HEETA and THETA.
Mechanisms of Leukotriene B4–Triggered Monocyte Adhesion
Authors: Friedrich Erik B. , Tager Andrew M. , Liu Emerson , Pettersson Annika , Owman Christer , Munn Lance , Luster Andrew D. , Gerszten Robert E.
Published: September 2003

Objective—Leukotriene B4(LTB4) has been implicated in the trafficking of monocytes to inflammatory pathologic conditions, such as transplant rejection and atherosclerosis. The aim of this study was to determine the mechanisms by which LTB4contributes to monocyte capture from the circulation.

Methods and Results—In in vitro and in vivo vascular models, the lipid chemoattractant LTB4was an equipotent agonist of monocyte adhesion compared with the chemokine monocyte chemoattractant protein-1 (MCP-1). Adenoviral gene transfer of specific endothelial adhesion molecules and blocking monoclonal antibody studies demonstrated that LTB4triggers both β1- and β2-integrin–dependent adhesion. Flow cytometry studies suggested that changes in integrin avidity or affinity, rather than alterations of integrin surface expression, were responsible for the chemoattractant-triggered arrest. Surprisingly, in contrast to the peptide chemokine MCP-1, LTB4did not activate the phosphoinositide 3-kinase pathway, which is a functionally critical step in chemokine-triggered effector functions.

Conclusions—LTB4is a potent trigger of monocyte adhesion under flow yet mediates its effects via pathways that appear to differ from peptide chemoattractants. A better understanding of the mechanisms of LTB4-induced monocyte trafficking might shed insight into disease pathogenesis and pinpoint critical steps for therapeutic intervention for multiple human inflammatory pathologic conditions.
Meta-Analysis of 4 Coronary Heart Disease Genome-Wide Linkage Studies Confirms a Susceptibility Locus on Chromosome 3q
Authors: Chiodini Benedetta D. , Lewis Cathryn M.
Published: September 2003

Objective—In coronary heart disease (CHD), 4 independent, genome-wide screens have now been published on Finnish, Mauritian, European, and Australian families. Results from these studies are inconclusive. We performed a meta-analysis to identify genetic regions that show evidence for susceptibility genes across studies.

Methods and Results—The rank-based genome-scan meta-analysis (GSMA) method was applied to the 4 CHD genome-wide linkage studies. The strongest evidence for linkage was found on chromosomes 3q26–27 (P=0.0001) and 2q34–37 (P=0.009). Analysis weighted by study size confirmed linkage in these regions (3q26–27,P=0.0002; 2q34–37,P=0.014).

Conclusions—The genetic regions 3q26–27 and 2q34–37 might contain susceptibility genes for CHD. Linkage to the 3q26-qter region has previously been shown in type 2 diabetes mellitus, metabolic syndrome, cholesterol concentration in LDL size fractions, and renal function in hypertensive subjects. The 2q34–37 region lies close to the type 2 diabetesNIDDM1locus. Both of these regions harbor several candidate genes involved in the homeostasis of glucose and lipid metabolism. These results are particularly intriguing, given the growing evidence of an association between CHD risk and metabolic abnormalities, such as insulin resistance, type 2 diabetes, abdominal obesity, and dyslipidemia.
Effects of Thrombin on Interactions Between β
3
-Integrins and Extracellular Matrix in Platelets and Vascular Cells
Authors: Stouffer G.A. , Smyth S.S.
Published: September 2003

The β
3
-integrin family consists of α
IIb
β
3
(also known as glycoprotein IIb/IIIa) and α
v
β
3
. α
IIb
β
3
is found on platelets and megakaryocytes and has an essential role in hemostasis. α
v
β
3
has a broader distribution, and it functions in angiogenesis, neointimal formation after vascular injury, and leukocyte trafficking. There are important interactions between thrombin and β
3
-integrins relative to both “inside-out” (integrin activation) and “outside-in” (modification of cellular events by ligand binding to integrins) signaling. Thrombin, by binding to G protein-coupled, protease-activated receptors, is a potent activator of α
IIb
β
3
. Conversely, outside-in signaling through α
IIb
β
3
amplifies events initiated by thrombin and is necessary for full platelet spreading, platelet aggregation, granule secretion, and the formation of a stable platelet thrombus. In smooth muscle cells, α
v
β
3
-integrins influence various responses to thrombin, including proliferation, c-Jun
NH
2
-terminal kinase-1 activation, and focal adhesion formation. Other interactions between β
3
-integrins and thrombin include β
3
-integrin promotion of the generation of thrombin by localizing prothrombin to cellular surfaces and/or enhancing the formation of procoagulant microparticles and the requirement of β
3
-integrin function for platelet-dependent clot retraction. In summary, there is increasing evidence that interactions between β
3
-integrins and thrombin play important roles in the regulation of hemostatic and vascular functions.
Inhibition of Tumor Necrosis Factor-α–Induced SHP-2 Phosphatase Activity by Shear Stress
Authors: Lerner-Marmarosh Nicole , Yoshizumi Masanori , Che Wenyi , Surapisitchat James , Kawakatsu Hisaaki , Akaike Masashi , Ding Bo , Huang Qunhua , Yan Chen , Berk Bradford C. , Abe Jun-ichi
Published: September 2003

Objectives—Atherosclerosis preferentially occurs in areas of turbulent flow, whereas laminar flow is atheroprotective. Inflammatory cytokines have been shown to stimulate adhesion molecule expression in endothelial cells that may promote atherosclerosis, in part, by stimulating c-JunN-terminal kinase (JNK) and nuclear factor (NF)-κB transcriptional activity.

Methods and Results—Because Src kinase family and Src homology region 2-domain phosphatase-2 (SHP-2) may regulate JNK activation, we studied the effect of shear stress on endothelial inflammation and JNK. Human umbilical vein endothelial cells preexposed to flow showed decreased tumor necrosis factor (TNF)-α–induced c-Jun and NF-κB transcriptional activation. TNF-α–mediated JNK, c-Jun, and NF-κB activation required Src and SHP-2 activity. Shear stress significantly inhibited SHP-2 phosphatase activity without affecting TNF-α–induced Src family kinase activation. Because MEKK3 and Gab1 are critical for TNF-α–induced c-Jun and NF-κB activation, we determined the role of SHP-2 phosphatase activity in MEKK3 signaling. A catalytically inactive form of SHP-2 increased MEKK3/Gab1 interaction and inhibited MEKK3 (but not MEKK1)-mediated c-Jun and NF-κB activation.

Conclusions—These results suggest that SHP-2 is a key mediator for the inhibitory effects of shear stress on TNF-α signaling in part via regulating MEKK3/Gab1 interaction, MEKK3 signaling, and subsequent adhesion molecule expression.
High Plasma Phospholipid Transfer Protein Levels as a Risk Factor for Coronary Artery Disease
Authors: Schlitt Axel , Bickel Christoph , Thumma Prathima , Blankenberg Stefan , Rupprecht Hans J. , Meyer Juergen , Jiang Xian-Cheng
Published: September 2003

Objective—

Plasma phospholipid transfer protein (PLTP) mediates both net transfer and exchange of phospholipids between different lipoproteins. Animal studies have shown that it is closely related to the development of atherosclerosis. PLTP-deficient mice have demonstrated increased antioxidation potential as well as a decrease in apolipoprotein B secretion and atherosclerotic lesions. In humans, high PLTP is associated with type II diabetes and obesity.

Methods and Results—

To assess the relationship between PLTP activity and coronary artery disease (CAD), a novel, high-throughput method to measure plasma PLTP activity was used, relating it to CAD in 1102 cases and 444 controls. This demonstrated that PLTP activity in patients with CAD was significantly higher than in controls (25.5 versus 22.4 pmol/μL per h;
P
<0.0001). Using multivariate logistic regression analysis, PLTP activity was found to have independent predictive value for CAD. Patients within the highest quintile of PLTP activity revealed a 1.9-fold increase in risk for CAD compared with patients within the lowest quintile.

Conclusions—

These findings indicate that PLTP activity is positively and independently related to CAD and suggest that (1) prospective studies to evaluate this relationship are warranted and (2) PLTP should be considered a therapeutic target.
Cardiovascular Risk Factors and Atherosclerosis in Young Males
Authors: Knoflach Michael , Kiechl Stefan , Kind Michaela , Said Michael , Sief Robert , Gisinger Martin , van der Zee Ruurd , Gaston Hill , Jarosch Elmer , Willeit Johann , Wick Georg
Published: September 2003

Background—

Necropsy studies suggest that atherosclerosis begins in childhood, but in vivo confirmation of this concept is sparse and limited to selected population samples. Furthermore, new risk concepts of atherosclerosis focusing on inflammation, infections, and immunity have not yet been evaluated in this age group.

Methods and Results—

This study was conducted in a sample of 141 17- to 18-year-old white males homogenous in age and sex. In addition to classic risk factors, C-reactive protein and the humoral and cellular immune reactivity to heat-shock proteins (HSPs) were assessed. Intima-media thickness (IMT) was quantified at 4 vessel segments of the carotid and femoral arteries. High IMT was considered to be present if the IMT of at least 1 vessel segment exceeded the 90th percentile. In a multivariate logistic regression analysis, cigarette smoking, high diastolic blood pressure, prominent immune reactivity to human and/or mycobacterial HSP60s, alcohol consumption (inverse), and low HDL cholesterol levels were all associated with high IMT. The prevalence of high IMT substantially increased from 0 to 60% when the number of risk conditions in a single individual increased from 0 to 4 (
P
<0.001 for linear trend).

Conclusions—

Our study supports the concept that atherosclerosis begins in the first decades of life and suggests a role of the immune system, especially immunoreactivity against HSP60s, in atherosclerosis of young individuals.
Mechanosensitive Cation Channels in Arterial Smooth Muscle Cells Are Activated by Diacylglycerol and Inhibited by Phospholipase C Inhibitor
Authors: Park Kyoung Sun , Kim Yangmi , Lee Young-Ho , Earm Yung E. , Ho Won-Kyung
Published: September 2003

Mechanosensitive cation channels may be involved in the development of the myogenic tone of arteries. The molecular identity of these channels is not clear, but transient receptor potential channels (TRPCs) are good candidates. In the present study, we searched for mechanosensitive channels at the single-channel level in arterial smooth muscle cells using the patch-clamp technique and investigated the channel properties in the light of properties of TRPCs. With 140 mmol/L CsCl in the pipette solution, application of negative pressures to the back of the pipette induced the activation of channels the open probability of which increased with the amount of negative pressure. The current-voltage relationship was linear in symmetrical ionic conditions, and the single-channel conductances for Cs
+
, K
+
, and Na
+
were 30, 36, and 27 pS, respectively. When NMDG
+
was substituted for Cs
+
in the pipette solution, inward currents were abolished, whereas outward currents remained active, indicating that the channels were nonselective to cations. The channel activity was blocked by intracellular Gd
3+
and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid and increased by diacylglycerol and by cyclopiazonic acid. Phospholipase C inhibitor (U73122) inhibited not only channel activity but also the development of myogenic tone induced by stretching of the basilar arteries. These results suggest that the ion channel responsible for the development of myogenic tone is the 30-pS mechanosensitive cation channel that exhibits properties similar to those of TRPCs.
Fas and Fas-Associated Death Domain Protein Regulate Monocyte Chemoattractant Protein-1 Expression by Human Smooth Muscle Cells Through Caspase- and Calpain-Dependent Release of Interleukin-1α
Authors: Schaub Friedemann J. , Liles W. Conrad , Ferri Nicola , Sayson Kirsten , Seifert Ronald A. , Bowen-Pope Daniel F.
Published: September 2003

We previously reported that treatment of human vascular smooth muscle cells (SMCs) with proapoptotic stimuli, including Fas ligand plus cycloheximide (FasL/Chx), or overexpression of Fas-associated death domain protein (FADD) result in increased expression of monocyte chemoattractant protein-1 (MCP-1) and other proinflammatory genes. In this study, we demonstrate that Fas/FADD-induced MCP-1 upregulation is driven by an autocrine/paracrine signaling loop in which interleukin (IL)-1α synthesis and release are activated through caspase- and calpain-dependent processes. Untreated SMCs contain very little IL-1α protein or transcript. Both were increased greatly in response to Fas/FADD activation, primarily through an autocrine/paracrine pathway in which secreted IL-1α stimulated additional IL-1α synthesis and release. Caspase 8 (Csp8) activity increased in response to FasL/Chx treatment, and Csp8 inhibitors markedly reduced IL-1α release and MCP-1 upregulation. In contrast, Csp8 activity was not significantly increased in response to FADD overexpression and caspase inhibitors did not effect FADD-induced MCP-1 upregulation. Both FasL/Chx treatment and FADD overexpression increased the activity of calpains. Calpain inhibitors reduced IL-1α release and MCP-1 upregulation in both FADD-overexpressing SMCs and FasL/Chx-treated SMCs without blocking Csp8 activity. This indicates that calpains are not required for activation of caspases and that caspase activation is not sufficient for IL-1α release and MCP-1 upregulation. These data suggest that calpains play a dominant role in Fas/FADD-induced IL-1α release and MCP-1 upregulation and that caspase activation may function to amplify the effects of calpain activation.
Sildenafil Prevents Change in RhoA Expression Induced by Chronic Hypoxia in Rat Pulmonary Artery
Authors: Sauzeau Vincent , Rolli-Derkinderen Malvyne , Lehoux Stéphanie , Loirand Gervaise , Pacaud Pierre
Published: September 2003

Exposure to chronic hypoxia (CH) induces a sustained pulmonary hypertension associated with structural and functional changes in the pulmonary arterial bed, including alterations of contractile properties. The small G-protein RhoA and its effector Rho kinase play a major role in the sustained rise in tension induced by vasoconstrictors. The aim of this study was to analyze the effect of CH on the RhoA/Rho kinase signaling pathway in the rat pulmonary artery. Maximal contraction of pulmonary artery rings to endothelin-1, noradrenaline, and the thromboxane A2 analog U46619 was markedly decreased in rats exposed to CH (10% O
2
, 2 weeks). This CH-induced decrease response to agonists was attributable to the abolition of RhoA-mediated Ca
2+
sensitization of the contraction. Real-time reverse transcriptase–polymerase chain reaction and Western blot analysis revealed a decrease in RhoA mRNA (79.4±6.0%, n=4) and RhoA (81.1±8.0%, n=4) expression in the main pulmonary artery from CH rats, whereas RhoA expression was not modified in arterial smooth muscle cells and arteries exposed to hypoxia and high intraluminal pressure, respectively. Treatment of rats with sildenafil (25 mg/kg per day) throughout 2 weeks of exposure to CH prevented CH-induced downregulation of RhoA, reduction of contraction, and pulmonary artery remodeling. These findings indicate that CH-induced downregulation of RhoA expression, leading to the abolition of RhoA/Rho kinase–mediated Ca
2+
sensitization of contraction, is responsible for the decreased responses to contracting agonists in the pulmonary artery of CH rats. These alterations are prevented by sildenafil, indicating a major role of the NO/cyclic GMP pathway in CH-induced altered RhoA signaling in the pulmonary artery.
Regulation of Connexin43 Gap Junctional Conductance by Ventricular Action Potentials
Authors: Lin Xianming , Crye Mark , Veenstra Richard D.
Published: September 2003

Transjunctional voltage regulates cardiac gap junctional conductance, but the kinetics of inactivation were considered too slow to affect cardiac action potential propagation. Connexin43 (Cx43) is abundantly expressed in the atrial and ventricular myocardium and the rapid ventricular conduction tissues (ie, His-Purkinje system) of the mammalian heart and is important to conduction through these cardiac tissues. The kinetics of Cx43 voltage gating were examined at peak action potential voltages using simulated ventricular myocardial action potential waveforms or pulse protocols exceeding 100-mV transjunctional potentials. Junctional current responses approximate the action potential morphology but conductance calculations reveal a 50% to 60% decline from peak to near constant plateau values. Junctional conductance recovers during phase 3 repolarization and early diastole to initial values. The bases for these transient changes in junctional conductance are the rapid decay kinetics in tens of milliseconds at peak transjunctional voltages (V
j
) of 130 mV and the gradual increase in junctional conductance as V
j
returns toward 0 mV. The decay time constants change e-fold per 22.1 mV above the half-inactivation voltage for Cx43 gap junctions of ±58 mV. A realistic dynamic model for changes in junctional resistance between excitable and nonexcitable cells during cardiac action potential propagation was developed based on these findings. This dynamic model of cardiac gap junctions will further our understanding of the role gap junctions play in the genesis and propagation of cardiac arrhythmias. The full text of this article is available online at http://www.circresaha.org.
Elevated Sarcoplasmic Reticulum Ca
2+
Leak in Intact Ventricular Myocytes From Rabbits in Heart Failure
Authors: Shannon Thomas R. , Pogwizd Steven M. , Bers Donald M.
Published: September 2003

Altered sarcoplasmic reticulum (SR) Ca
2+
-ATPase and Na
+
-Ca
2+
exchange (NCX) function have been implicated in depressing SR Ca
2+
content and contractile function in heart failure (HF). Enhanced diastolic ryanodine receptor (RyR) leak could also lower SR Ca
2+
load in HF, but direct cellular measurements are lacking. In this study, we measure SR Ca
2+
leak directly in intact isolated rabbit ventricular myocytes from a well-developed nonischemic HF model. Abrupt block of SR Ca
2+
leak by tetracaine shifts Ca
2+
from the cytosol to SR. The tetracaine-induced decline in [Ca
2+
]
i
and increase total SR Ca
2+
load ([Ca
2+
]
SRT
) directly indicate the SR Ca
2+
leak (before tetracaine). Diastolic SR Ca
2+
leak increases with [Ca
2+
]
SRT
, and for any [Ca
2+
]
SRT
is greater in HF versus control. Mathematical modeling was used to compare the relative impact of alterations in SR Ca
2+
leak, SR Ca
2+
-ATPase, and Na
+
-Ca
2+
exchange on SR Ca
2+
load in HF. We conclude that increased diastolic SR Ca
2+
leak in HF may contribute to reductions in SR Ca
2+
content, but changes in NCX in this HF model have more impact on [Ca
2+
]
SRT
.