bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and functional characterization of the human GLUT7 isoform SLC2A7 from the small intestine
Authors: Li Qiang , Manolescu Andrei , Ritzel Mabel , Yao Sylvia , Slugoski Melissa , Young James D. , Chen Xing-Zhen , Cheeseman Chris I.
Published: March 2004

Facilitated glucose transporters (GLUTs) mediate transport of sugars across cell membranes by using the chemical gradient of sugars as the driving force. Improved cloning techniques and database analyses have expanded this family of proteins to a total of 14 putative members. In this work a novel hexose transporter isoform, GLUT7, has been cloned from a human intestinal cDNA library by using a PCR-based strategy (GenBank accession no. AY571960 ). The encoded protein is comprised of 524 amino acid residues and shares 68% similarity and 53% identity with GLUT5, its most closely related isoform. When GLUT7 was expressed in Xenopus oocytes, it showed high-affinity transport for glucose ( Km= 0.3 mM) and fructose (IC50= 0.060 mM). Galactose, 2-deoxy-d-glucose, and xylose were not transported. Uptake of 100 μM d-glucose was not inhibited by 200 μM phloretin or 100 μM cytochalasin B. Northern blotting indicated that the mRNA for GLUT7 is present in the human small intestine, colon, testis, and prostate. Western blotting and immunohistochemistry of rat tissues with an antibody raised against the predicted COOH-terminal sequence confirmed expression of the protein in the small intestine and indicated that the transporter is predominantly expressed in the enterocytes' brush-border membrane. The unusual substrate specificity and close sequence identity with GLUT5 suggest that GLUT7 represents an intermediate between class II GLUTs and the class I member GLUT2. Comparison between these proteins may provide key information as to the structural determinants for the recognition of fructose as a substrate.
α-Tropomyosin mutations Asp175Asn and Glu180Gly affect cardiac function in transgenic rats in different ways
Authors: Wernicke Dirk , Thiel Corinna , Duja-Isac Corina M. , Essin Kirill V. , Spindler Matthias , Nunez Derek J. R. , Plehm Ralph , Wessel Niels , Hammes Annette , Edwards Robert-J. , Lippoldt Andrea , Zacharias Ute , Strömer Hinrik , Neubauer Stefan , Davies Michael J. , Morano Ingo , Thierfelder Ludwig
Published: March 2004

To study the mechanisms by which missense mutations in α-tropomyosin cause familial hypertrophic cardiomyopathy, we generated transgenic rats overexpressing α-tropomyosin with one of two disease-causing mutations, Asp175Asn or Glu180Gly, and analyzed phenotypic changes at molecular, morphological, and physiological levels. The transgenic proteins were stably integrated into the sarcomere, as shown by immunohistochemistry using a human-specific anti-α-tropomyosin antibody, ARG1. In transgenic rats with either α-tropomyosin mutation, molecular markers of cardiac hypertrophy were induced. Ca2+sensitivity of cardiac skinned-fiber preparations from animals with mutation Asp175Asn, but not Glu180Gly, was decreased. Furthermore, elevated frequency and amplitude of spontaneous Ca2+waves were detected only in cardiomyocytes from animals with mutation Asp175Asn, suggesting an increase in intracellular Ca2+concentration compensating for the reduced Ca2+sensitivity of isometric force generation. Accordingly, in Langendorff-perfused heart preparations, myocardial contraction and relaxation were accelerated in animals with mutation Asp175Asn. The results allow us to propose a hypothesis of the pathogenetic changes caused by α-tropomyosin mutation Asp175Asn in familial hypertrophic cardiomyopathy on the basis of changes in Ca2+handling as a sensitive mechanism to compensate for alterations in sarcomeric structure.
Inhibition of the formation of EETs and 20-HETE with 1-aminobenzotriazole attenuates pressure natriuresis
Authors: Dos Santos Elisabete A. , Dahly-Vernon Annette J. , Hoagland Kimberly M. , Roman Richard J.
Published: March 2004

This study examined the effects of chronic blockade of the renal formation of epoxyeicosatrienoic acids and 20-hydroxyeicosatetraenoic acid with 1-aminobenzotriazole (ABT; 50 mg·kg−1· day−1ip for 5 days) on pressure natriuresis and the inhibitory effects of elevations in renal perfusion pressure (RPP) on Na+-K+-ATPase activity and the distribution of the sodium/hydrogen exchanger (NHE)-3 in the proximal tubule of rats. In control rats ( n = 15), sodium excretion rose from 2.3 ± 0.4 to 19.4 ± 1.8 μeq·min−1·g kidney weight−1when RPP was increased from 114 ± 1 to 156 ± 2 mmHg. Fractional excretion of lithium rose from 28 ± 3 to 43 ± 3% of the filtered load. Chronic treatment of the rats with ABT for 5 days ( n = 8) blunted the natriuretic response to elevations in RPP by 75% and attenuated the increase in fractional excretion of lithium by 45%. In vehicle-treated rats, renal Na+-K+-ATPase activity fell from 31 ± 5 to 19 ± 2 μmol Pi·mg protein−1·h−1and NHE-3 protein was internalized from the brush border of the proximal tubule after an elevation in RPP. In contrast, Na+-K+-ATPase activity and the distribution of NHE-3 protein remained unaltered in rats treated with ABT. These results suggest that cytochrome P-450 metabolites of arachidonic acid contribute to pressure natriuresis by inhibiting Na+-K+-ATPase activity and promoting internalization of NHE-3 protein from the brush border of the proximal tubule.
An apical K+-dependent HCO3− transport pathway opposes transepithelial HCO3− absorption in rat medullary thick ascending limb
Authors: Watts Bruns A. , Good David W.
Published: March 2004

Absorption of HCO3− in the medullary thick ascending limb (MTAL) is mediated by apical membrane Na+/H+ exchange. The identity and function of other apical acid-base transporters in this segment have not been defined. The present study was designed to examine apical membrane HCO3−/OH−/H+ transport pathways in the rat MTAL and to determine their role in transepithelial HCO3− absorption. MTALs were perfused in vitro in Na+- and Cl−-free solutions containing 25 mM HCO3−, 5% CO2. Lumen addition of either 120 mM Cl− or 50 mM Na+ (50 μM EIPA present) had no effect on intracellular pH (pHi). Lumen Cl− addition also had no effect on pHi in the presence of 145 mM Na+ or in the nominal absence of HCO3−/CO2. Thus there was no evidence for apical Cl−/HCO3− (OH−) exchange, Na+-dependent Cl−/HCO3− exchange, or Na+-HCO3− cotransport. In contrast, in tubules studied in Na+- and Cl−-free solutions containing 25 mM HCO3−, 5% CO2 and 120 mM K+, removal of luminal K+ induced a rapid and pronounced decrease in pHi (ΔpHi = 0.56 ± 0.06 pH U). pHi recovered following lumen K+ readdition. The initial rate of net base efflux induced by lumen K+ removal was decreased 85% at the same pHi in the nominal absence of HCO3−/CO2, indicating a dependence on HCO3−/CO2 and arguing against apical K+/H+ exchange. A combination of the apical K+ channel blockers quinidine (0.1 mM) and glybenclamide (0.25 mM) had no effect on the lumen K+-induced pHi changes, arguing against electrically coupled K+ and HCO3− conductances. The effect of lumen K+ on pHi was inhibited by 1 mM H2DIDS. In addition, lumen addition of DIDS increased transepithelial HCO3− absorption from 10.7 ± 0.7 to 14.9 ± 0.7 pmol·min−1·mm−1 ( P < 0.001) and increased pHi slightly in MTAL studied in physiological solutions (25 mM HCO3− and 4 mM K+). Lumen DIDS stimulated HCO3− absorption in the absence and presence of furosemide. These results are consistent with an apical membrane K+-dependent HCO3− transport pathway that mediates coupled transfer of K+ and HCO3− from cell to lumen in the MTAL. This mechanism, possibly an apical K+-HCO3− cotransporter, functions in parallel with apical Na+/H+ exchange and opposes transepithelial HCO3− absorption.
Intracerebroventricular physostigmine facilitates heat loss mechanisms in running rats
Authors: Rodrigues Alex G. , Lima Nilo R. V. , Coimbra Cândido C. , Marubayashi Umeko
Published: March 2004

The aim of this study was to evaluate the participation of central cholinergic transmission in the regulation of metabolic rate, core temperature, and heat storage in untrained rats submitted to exercise on a treadmill (20 m/min, 5% inclination) until fatigue. The animals were separated into eight experimental groups, and core temperature or metabolic rate was measured in the rats while they were exercising or while they were at rest after injection of 2 μl of 5 × 10−3 M physostigmine (Phy) or 0.15 M NaCl solution (Sal) into the lateral cerebral ventricle. Metabolic rate was determined by the indirect calorimetry system, and colonic temperature was recorded as an index of core temperature. In resting animals, Phy induced only a small increase in metabolic rate compared with Sal injection, without having any effect on core temperature. During exercise, the Phy-treated animals showed a lower core heating rate (0.022 ± 0.003°C/min Phy vs. 0.033 ± 0.003°C/min Sal; P < 0.02), lower heat storage (285 ± 37 cal Phy vs. 436 ± 34 cal Sal; P < 0.02) and lower core temperature at fatigue point than the Sal-treated group (38.5 ± 0.1°C Phy vs. 39.0 ± 0.1°C Sal; P < 0.05). However, despite the lower core heating rate, heat storage, and core temperature at fatigue, the Phy-treated rats showed a similar running time compared with the Sal-treated group. We conclude that the activation of the central cholinergic system during exercise increases heat dissipation and attenuates the exercise-induced increase in core temperature without affecting running performance.
Longitudinal changes in the kinetic response to heavy-intensity exercise in children
Authors: Fawkner Samantha G. , Armstrong Neil
Published: March 2004

The purpose of this study was to investigate longitudinal changes with age in the kinetic response to cycling at heavy-intensity exercise in boys and girls. Twenty-two prepubertal children (13 male, 9 female) carried out a series of exercise tests on two test occasions with a 2-yr interval. On each test occasion, the subject completed multiple transitions from baseline to 40% of the difference between their previously determined V-slope and peak O2 uptake (V̇o2) for 9 min on an electronically braked cycle ergometer. Each subject's breath-by-breath responses were interpolated to 1-s intervals, time aligned, and averaged. The data after phase 1 were fit with 1) a double exponential model and 2) a single exponential model within a fitting window that was previously identified to exclude the slow component. There were no significant differences in the parameters of the primary component between each model. Subsequent analysis was carried out using model 2. The V̇o2 slow component was computed as the difference between the amplitude of the primary component and the end-exercise V̇o2 and was expressed as the percent contribution to the total change in V̇o2. Over the 2-yr period, the primary time constant (boys 16.8 ± 5.3 and 21.7 ± 5.3 s, girls 21.1 ± 8.1 and 26.4 ± 8.4 s, first and second occasion, respectively) and the relative amplitude of the slow component (boys 9.4 ± 4.6 and 13.8 ± 5.3%, girls 10.3 ± 2.4 and 15.5 ± 2.8%, first and second occasion, respectively) significantly increased with no sex differences. The data demonstrate that children do display a slow-component response to exercise and are consistent with an age-dependent change in the muscles' potential for O2 utilization.
Unrestrained plethysmography is an unreliable measure of airway responsiveness in BALB/c and C57BL/6 mice
Authors: Adler Andy , Cieslewicz Greg , Irvin Charles G.
Published: March 2004

There has been significant utilization of the technique described by Hamelmann et al. ( Am J Respir Crit Care Med 156: 766–775, 1997) in which a parameter, enhanced pause (Penh), related to airways responsiveness is noninvasively measured by unrestrained plethysmography (UP). Investigating this technique, we sought to answer these questions: 1) How do changes in Penh compare with changes in traditional plethysmographic and lung mechanical parameters? 2) How do UP parameters perform in two different mouse strains? Awake immunized and control BALB/c ( n = 16) and C57BL/6 ( n = 14) mice were placed in the UP chamber and exposed to doses of aerosolized methacholine while the following parameters were measured at each concentration: inspiratory time (Ti), expiratory time (Te), total time (Ttot), Ti/Ttot, peak inspiratory pressure, peak expiratory pressure, Pause, Penh, tidal volume (Vt), Vt/Ti, Vt/Te, and Vt/Ttot. The next day, lung resistance (Rl) and compliance (Cl) were invasively measured in the same animals. For the BALB/c, the parameters with the highest magnitude of correlation coefficient vs. Rl are (in order) 1) Cl, 2) Pause and Penh, 3) parameters of breathing frequency (Te, Ttot, Ti), and 4) parameters related to Vt (inspiratory pressure, expiratory pressure). Flow parameters (Vt/Ttot, Vt/Te, Vt/Ti) and duty cycle parameters (Ti/Ttot) had insignificant correlations. This ordering is significantly different in C57BL/6 mice, in which the parameters with the largest correlations are 1) Cl, 2) parameters of breathing frequency, and 3) flow parameters. Pause, Penh, Vt, and duty cycle parameters had insignificant correlations. These data show that Penh is problematic in the sense that it is strain specific; it behaves very differently in BALB/c and C57BL/6 mice. We suggest that UP parameters largely originate as part of reflex control of breathing processes, rather than in the lung mechanics and conclude that it is inappropriate to use UP parameters in general, and Penh specifically, as substitute variables for invasive mechanical indexes such as Rl.
5-HT offsets homeostasis of synaptic transmission during short-term facilitation
Authors: Sparks G. M. , Cooper R. L.
Published: March 2004

In this study, we approach the topic of vesicle recruitment and recycling by perturbing neurotransmission at the crayfish neuromuscular junction with altered electrical activity and the presence of the neuromodulator serotonin (5-HT). After induction of short-term facilitation (STF) with stimulus pulse trains (40 Hz, 20 pulses), the amount of synaptic transmission can be maintained at a relatively constant level, producing a plateau in the amplitude of the excitatory postsynaptic potentials (EPSPs) throughout the remaining stimuli within a train of a few hundred milliseconds. With an increase in the frequency of the stimuli within a train (60 Hz, 20 pulses), an altered plateau of larger EPSP amplitudes occurs. This suggests that differential rates of vesicle recruitment can be rapidly reached and maintained. Exposure of nerve terminals to 5-HT further enhances the EPSP amplitudes to yet a higher plateau level. The effect of 5-HT is more pronounced for 40-Hz pulse trains than for 60-Hz trains. This suggests that 5-HT can recruit vesicles into the readily releasable pool (RRP) and that the recruitment is limited at higher stimulation frequencies. The attainment of a larger amplitude in the plateaus of the EPSPs at 60 Hz compared with 40 Hz also suggests that the rapid induction of STF enhances the entry of vesicles into the RRP. By direct quantal counts, mean quantal content increases linearly during STF, and 5-HT offsets the linear release. We propose that 5-HT and electrically induced recruitment of vesicles from a reserve pool to the RRP may share similar recruitment mechanisms.
Encoder Adaptation Modulates the Visual Responses of Crayfish Interneurons
Authors: Glantz Raymon M. , Schroeter John P.
Published: March 2004

The responses of sustaining and dimming fibers were characterized by the time varying firing rates elicited by extrinsic current and flashes of light. These data were simulated by an adaptive integrate-and-fire model. A postimpulse shunt conductance simulated spike-frequency adaptation. The correlation between observed and model current-elicited impulse rates was 0.94–0.98. However, except for a difference in input resistance (both measured and simulated), the voltage to impulse encoders of the two cell groups was similar and exhibited comparable degrees of spike-frequency adaptation (40 to 45%). The encoder model derived from current-elicited responses (with fixed parameters) was used to simulate visual responses elicited by light flashes. These simulations included a synaptic current derived from the time course of the postsynaptic potential (PSP). The sustaining fiber visual response consisted of a large excitatory PSP and high-frequency transient burst that adapted (by ∼80%) to a low-frequency plateau discharge. The simulations indicated that spike-frequency adaptation had no effect on the transient discharge but reduced the plateau firing rate by ∼60%. Encoder adaptation enhances the sustaining fiber response to the time derivative of the stimulus. In dimming fibers, the light flash elicits an inhibitory PSP that interrupts the “dark discharge” and an off response following the end of the flash. The simulations indicated that spike-frequency adaptation reduces the firing rate of both the dark discharge and the off response. Thus the model suggests that different effects of encoder adaptation on the two cell types arise from the same encoder mechanisms, but different actions are determined by differences in impulse rate and the time course of the discharge.
Activation of Monkey Locus Coeruleus Neurons Varies With Difficulty and Performance in a Target Detection Task
Authors: Rajkowski Janusz , Majczynski Henryk , Clayton Edwin , Aston-Jones Gary
Published: March 2004

We previously reported that noradrenergic neurons in the monkey locus coeruleus (LC) are activated selectively by target stimuli in a target detection task. Here, we varied the discrimination difficulty in this task and recorded impulse activity of LC neurons to analyze LC responses on error trials and in relation to behavioral response times (RTs). In easy and difficult discrimination conditions, LC neurons responded preferentially to target stimuli with phasic activation. These responses consistently preceded behavioral responses regardless of task difficulty. Latencies for LC and behavioral responses increased similarly for difficult compared with easy discrimination trials. LC response latencies were also shorter for fast RT trials compared with slow RT trials regardless of difficulty, indicating a close temporal relationship between LC and behavioral responses. This relationship was confirmed with response-locked histograms of LC activity, which yielded more temporally synchronized LC responses than stimulus-locked histograms. Population histograms of LC activity revealed that nontarget stimuli resulting in false alarm responses produced phasic LC activation (although smaller than for target-hit trials), and nontarget stimuli resulting in correct rejection responses yielded a small inhibition in LC activity. Population analyses also revealed that LC responses included an early, small excitatory component that was not previously detected. This early response was nondiscriminative because it was similar for target and nontarget stimulus trials. These results indicate that LC neurons exhibit early small magnitude responses that are closely linked to sensory stimuli. In addition, these cells show a later, larger magnitude response that is temporally linked to behavioral responses. These and other results lead us to hypothesize that LC responses are driven by decision processes and help facilitate subsequent behavioral responses.
Molecular Basis for Modulation of Recombinant α1β2γ2 GABAA Receptors by Protons
Authors: Huang Ren-Qi , Chen Zhenglan , Dillon Glenn H.
Published: March 2004

We have previously shown that extracellular protons inhibit recombinant and native GABAA receptors. In this report, we studied the site(s) and mechanism by which protons modulate the GABAA receptor. Whole cell GABA-activated currents were recorded from human embryonic kidney (HEK) 293 cells expressing recombinant α1β2γ2 GABAA receptors. Protons competitively inhibited the response to GABA and bicuculline. In contrast, change in pH did not influence direct gating of the channel by pentobarbital, and it did not influence spontaneous channel openings in α1(L264T)β2γ2 receptors, suggesting pH does not modulate channel activity by affecting the channel gating process directly. To test the hypothesis that protons modulate GABAA receptors at the ligand binding site, we systemically mutated N-terminal residues known to be involved in GABA binding and assessed effects of pH on these mutant receptors. Site-specific mutation of β2 Y205 to F or α1 F64 to A, both of which are known to influence GABA binding, significantly reduced pH sensitivity of the GABA response. These mutations did not affect Zn2+ sensitivity, suggesting that H+ and Zn2+ do not share a common site of action. Additional experiments further tested this possibility. Treatment with the histidine-modifying reagent diethylpyrocarbonate (DEPC) reduced Zn2+-mediated inhibition of GABAA receptors but had no effect on proton-induced inhibition of GABA currents. In addition, mutation of residues known to be involved in Zn2+ modulation had no effect on pH modulation of GABAA receptors. Our results support the hypothesis that protons inhibit GABAA receptor function by direct or allosteric interaction with the GABA binding site. In addition, the sites of action of H+ and Zn2+ in GABAA receptors are distinct.
Tissue Angiotensin-Converting Enzyme in Imposed and Physiological Flow-Related Arterial Remodeling in Mice
Authors: Hilgers Rob H.P. , Schiffers Paul M.H. , Aartsen Wendy M. , Fazzi Gregorio E. , Smits Jos F.M. , De Mey Jo G.R.
Published: March 2004

Objective—

To test whether membrane-bound angiotensin I-converting enzyme (t-ACE) is involved in arterial remodeling, we applied unilateral carotid artery (CA) ligation and studied uterine arteries (UA) before, during, and after pregnancy in t-ACE
−/−
and t-ACE
+/+
mice.

Results—

In CA of t-ACE
−/−
mice, blood pressure, outer diameter (D), and medial cross-sectional area (mCSA) were reduced, whereas blood flow (BF) and the number of medial cells (mC) were not modified. In the ligated CA, mCSA and number of mC were increased while outer D and distensibility were reduced. These changes were significantly less pronounced in t-ACE
−/−
than t-ACE
+/+
mice. In UA of t-ACE
−/−
mice, D was larger and mCSA was unaltered. At term pregnancy, D and mCSA of the UA were reversibly increased. Structural changes of UA during and after pregnancy were comparable in both strains.

Conclusions—

t-ACE contributes to arterial structure and remodeling. It plays a major role in hyperplastic inward remodeling of the CA imposed by blood flow cessation, but it is not essential for outward hypertrophic and subsequent inward hypotrophic remodeling of the UA during and after pregnancy.
High Glucose–Induced Alterations in Subendothelial Matrix Perlecan Leads to Increased Monocyte Binding
Authors: Vogl-Willis Catherine A. , Edwards Iris J.
Published: March 2004

Objective—Hyperglycemia is an independent risk factor for cardiovascular disease in diabetic patients, although the link between the two is unknown. These studies were designed to model effects of high glucose on an early event in atherogenesis: the binding of monocytes to subendothelial matrix (SEM).

Methods and Results—SEM was prepared from human bovine aortic endothelial cells (HAECs) and bovine aortic endothelial cells (BAECs) cultured in the presence of low (5 mmol/L) or high (30 mmol/L) glucose for 3 to 5 days. Monocyte binding was significantly higher (P<0.05) to the SEM of both HAEC and BAEC exposed to high glucose. This increase was a result of changes in SEM heparan sulfate proteoglycans (HSPGs). Metabolic radiolabeling of BAEC demonstrated a 24% decrease in [35S]sulfate incorporation into SEM HSPG produced by cells incubated in 30 mmol/L versus 5 mmol/L glucose, whereas no glucose-associated differences were measured in [35S]methionine incorporation into proteoglycans (PGs) or non-PG proteins. Autoradiography revealed 2 high-molecular weight SEM HSPGs. One was a hybrid PG that contained both heparan sulfate and chondroitin sulfate/dermatan sulfate chains. Both PGs were identified by Western blotting as perlecan.

Conclusions—These results illustrate that hyperglycemia-induced structural changes in perlecan may result in a SEM that is more favorable to retention of monocytes.
Lower Loop Reentry as a Mechanism of Clockwise Right Atrial Flutter
Authors: Zhang Shulong , Younis George , Hariharan Ramesh , Ho John , Yang Yanfei , Ip John , Thakur Ranjun K. , Seger John , Scheinman Melvin M. , Cheng Jie
Published: March 2004

Background—

Right atrial reentrant tachycardia resulting from lower loop reentry (LLR) around the inferior vena cava (IVC) has been described recently. However, all reported cases of LLR in the literature have negative flutter waves on the inferior surface ECG leads similar to that of counterclockwise typical atrial flutter around the tricuspid annulus (TA). Right atrial flutter with positive flutter waves in the inferior ECG leads has been assumed to rotate as a single reentrant activation wave front around the TA, and the role of LLR in those patients is not known.

Methods and Results—

Twelve consecutive patients with flutter wave morphology on surface ECG consistent with clockwise atrial flutter were studied. The endocardial activation pattern recorded from conventional multipolar electrode catheters was characteristic of clockwise atrial flutter around the TA. Entrainment pacing in all 12 patients and 3D activation sequence mapping in 7 patients, however, revealed clockwise LLR involving the lower right atrium around the IVC in 7 patients, figure-of-8 double-loop reentry around both the IVC and TA in 4, and single reentrant loop around the TA in 1. Linear radiofrequency ablation in the isthmus between the TA and IVC (TI isthmus) terminated the tachycardia in all patients.

Conclusions—

Surface ECG flutter wave morphology and limited recording intracardiac sites proved insufficient to delineate the precise mechanism of the TI isthmus–dependent clockwise right atrial flutters. Most right atrial flutters with positive flutter wave on surface ECG may be supported by a reentrant circuit around the IVC or a figure-of-8 double-loop reentry involving both the IVC and TA.
Possible Protective Role for C-Reactive Protein in Atherogenesis
Authors: Bhakdi Sucharit , Torzewski Michael , Paprotka Kerstin , Schmitt Steffen , Barsoom Hala , Suriyaphol Prapat , Han Shan-Rui , Lackner Karl J. , Husmann Matthias
Published: March 2004

Background—

Previous work indicated that enzymatically remodeled LDL (E-LDL) might activate complement in atherosclerotic lesions via a C-reactive protein (CRP)–dependent and CRP-independent pathway. We sought to substantiate this contention and determine whether both pathways drive the sequence to completion.

Methods and Results—

E-LDL was prepared by sequential treatment of LDL with a protease and cholesteryl esterase. Trypsin, proteinase K, cathepsin H, or plasmin was used with similar results. Functional tests were used to assess total complement hemolytic activity, and immunoassays were used to demonstrate C3 cleavage and to quantify C3a, C4a, C5a, and C5b-9. E-LDL preparations activated complement to completion, independent of CRP, when present above a threshold concentration (100 to 200 μg/mL in 5% serum). Below the threshold, all E-LDL preparations activated complement in dependence of CRP, but the pathway then halted before the terminal sequence. Native LDL and oxidized LDL did not activate complement under any circumstances tested. Immunohistological analyses corroborated the concept that CRP-dependent complement activation inefficiently generates C5b-9.

Conclusions—

Binding of CRP to E-LDL is the first trigger for complement activation in the atherosclerotic lesion, but the terminal sequence is thereby spared. This putatively protective function of CRP is overrun at higher E-LDL concentrations, so that potentially harmful C5b-9 complexes are generated.
Profoundly Reduced Neovascularization Capacity of Bone Marrow Mononuclear Cells Derived From Patients With Chronic Ischemic Heart Disease
Authors: Heeschen Christopher , Lehmann Ralf , Honold Jörg , Assmus Birgit , Aicher Alexandra , Walter Dirk H. , Martin Hans , Zeiher Andreas M. , Dimmeler Stefanie
Published: March 2004

Background—

Cell therapy with bone marrow–derived stem/progenitor cells is a novel option for improving neovascularization and cardiac function in ischemic heart disease. Circulating endothelial progenitor cells in patients with coronary heart disease are impaired with respect to number and functional activity. However, whether this impairment also extends to bone marrow–derived mononuclear cells (BM-MNCs) in patients with chronic ischemic cardiomyopathy (ICMP) is unclear.

Methods and Results—

BM-MNCs were isolated from bone marrow aspirates in 18 patients with ICMP (ejection fraction, 38±11%) and 8 healthy control subjects (controls). The number of hematopoietic stem/progenitor cells (CD34
+
/CD133
+
), CD49d
+
(VLA-4) cells, and CXCR4
+
cells did not differ between the 2 groups. However, the colony-forming capacity of BM-MNCs from patients with ICMP was significantly lower compared with BM-MNCs from healthy controls (37.3±25.0 versus 113.8±70.4 granulocyte-macrophage colony-forming units;
P
=0.009). Likewise, the migratory response to stromal cell–derived factor 1 (SDF-1) and vascular endothelial growth factor (VEGF) was significantly reduced in BM-MNCs derived from patients with ICMP compared with BM-MNCs from healthy controls (SDF-1, 46.3±26.2 versus 108.6±40.4 cells/microscopic field,
P
<0.001; VEGF, 34±24.2 versus 54.8±29.3 cells/microscopic field,
P
=0.027). To assess the in vivo relevance of these findings, we tested the functional activity of BM-MNCs to improve neovascularization in a hindlimb animal model using nude mice. Two weeks after ligation of the femoral artery and intravenous injection of 5×10
5
BM-MNCs, laser Doppler–derived relative limb blood flow in mice treated with BM-MNCs from patients with ICMP was significantly lower compared with mice treated with BM-MNCs from healthy controls (0.45±0.14 versus 0.68±0.15;
P
<0.001). The in vivo neovascularization capacity of BM-MNCs closely correlated with the in vitro assessment of SDF-1–induced migration (
r
=0.78;
P
<0.001) and colony-forming capacity (
r
=0.74;
P
<0.001).

Conclusions—

BM-MNCs isolated from patients with ICMP have a significantly reduced migratory and colony-forming activity in vitro and a reduced neovascularization capacity in vivo despite similar content of hematopoietic stem cells. This functional impairment of BM-MNCs from patients with ICMP may limit their therapeutic potential for clinical cell therapy.
Clinical Pharmacology of Platelet, Monocyte, and Vascular Cyclooxygenase Inhibition by Naproxen and Low-Dose Aspirin in Healthy Subjects
Authors: Capone Marta L. , Tacconelli Stefania , Sciulli Maria G. , Grana Marilena , Ricciotti Emanuela , Minuz Pietro , Di Gregorio Patrizia , Merciaro Gabriele , Patrono Carlo , Patrignani Paola
Published: March 2004

Background—

The current controversy on the potential cardioprotective effect of naproxen prompted us to evaluate the extent and duration of platelet, monocyte, and vascular cyclooxygenase (COX) inhibition by naproxen compared with low-dose aspirin.

Methods and Results—

We performed a crossover, open-label study of low-dose aspirin (100 mg/d) or naproxen (500 mg BID) administered to 9 healthy subjects for 6 days. The effects on thromboxane (TX) and prostacyclin biosynthesis were assessed up to 24 hours after oral dosing. Serum TXB
2
, plasma prostaglandin (PG) E
2
, and urinary 11-dehydro-TXB
2
and 2,3-dinor-6-keto-PGF
1α
were measured by previously validated radioimmunoassays. The administration of naproxen or aspirin caused a similar suppression of whole-blood TXB
2
production, an index of platelet COX-1 activity ex vivo, by 94±3% and 99±0.3% (mean±SD), respectively, and of the urinary excretion of 11-dehydro-TXB
2
, an index of systemic biosynthesis of TXA
2
in vivo, by 85±8% and 78±7%, respectively, that persisted throughout the dosing interval. Naproxen, in contrast to aspirin, significantly reduced systemic prostacyclin biosynthesis by 77±19%, consistent with differential inhibition of monocyte COX-2 activity measured ex vivo.

Conclusions—

The regular administration of naproxen 500 mg BID can mimic the antiplatelet COX-1 effect of low-dose aspirin. Naproxen, unlike aspirin, decreased prostacyclin biosynthesis in vivo.
Association of Type 2 Diabetes With Cyclooxygenase-Mediated Inflammation and Oxidative Stress in an Elderly Population
Authors: Helmersson Johanna , Vessby Bengt , Larsson Anders , Basu Samar
Published: March 2004

Background—

Involvement of cyclooxygenase (COX)-mediated inflammation in type 2 diabetes has not been studied, and the association between cytokine-mediated inflammation and diabetes is not fully clarified.

Methods and Results—

15-Keto-dihydro-prostaglandin F
2α
(a metabolite of prostaglandin F
2α
and an indicator of COX-mediated inflammation), high-sensitivity C-reactive protein (CRP), serum amyloid protein A (SAA), 8-iso-PGF
2α
(a nonenzymatic, free radical product of arachidonic acid and an indicator of oxidative stress), and α-tocopherol were measured in a population-based sample of 77-year-old men (n=765), in which 112 men had type 2 diabetes. The inflammatory indicators were increased in men with diabetes (urinary 15-keto-dihydro-PGF
2α
,
P
<0.001, CRP and SAA,
P
<0.05). However, when adjusted for body mass index, waist circumference, or fasting insulin, no association was found between diabetes and CRP or SAA. The oxidative stress indicator 8-iso-PGF
2α
in urine was increased (
P
<0.01) in men with diabetes. Patients who were newly diagnosed with diabetes (<7 years since diagnosis) had increased urinary 15-keto-dihydro-PGF
2α
and decreased α-tocopherol, but 8-iso-PGF
2α
was unaltered.

Conclusions—

This is the first study to show that type 2 diabetes in elderly men is related to COX-mediated inflammation, reflected by enhanced prostaglandin formation. The high levels of cytokine-mediated acute-phase proteins observed in men with diabetes appear to be related to obesity and increased fasting insulin. The results further suggest that the appearance of chronic inflammation is an early process in the pathogenesis of diabetes, whereas oxidative injury may be a later process, possibly related to inflammation.
Effects of Acute Mental Stress and Exercise on T-Wave Alternans in Patients With Implantable Cardioverter Defibrillators and Controls
Authors: Kop Willem J. , Krantz David S. , Nearing Bruce D. , Gottdiener John S. , Quigley John F. , O’Callahan Mark , DelNegro Albert A. , Friehling Ted D. , Karasik Pamela , Suchday Sonia , Levine Joseph , Verrier Richard L.
Published: March 2004

Background—

Malignant cardiac arrhythmias can be triggered by exercise and by mental stress in vulnerable patients. Exercise-induced T-wave alternans (TWA) is an established marker of cardiac electrical instability. However, the effects of acute mental stress on TWA have not been investigated as a vulnerability marker in humans.

Methods and Results—

TWA responses to mental stress (anger recall and mental arithmetic) and bicycle ergometry were evaluated in patients with implantable cardioverter defibrillators (ICDs) and documented coronary artery disease (n=23, age 62.1±12.3 years) and controls (n=17, age 54.2±12.1 years). TWA was assessed from digitized ECGs by modified moving average analysis. Dual-isotope single photon emission computed tomography was used to assess myocardial ischemia. TWA increased during mental stress and exercise (
P
values <0.001), and TWA responses were higher in ICD patients than in controls (arithmetic Δ=8.9±1.4 versus 4.3±2.2 μV,
P
=0.043; exercise Δ=21.4±2.8 versus 13.8±3.2 μV,
P
=0.038). TWA increases with mental stress occurred at substantially lower heart rates (anger recall Δ=9.7±7.7 bpm, arithmetic Δ=14.3±13.3 bpm) versus exercise (Δ=53.7±22.7 bpm;
P
values <0.001). After adjustment for heart rate increases, mental stress and exercise provoked increased TWA in ICD patients (
P
values <0.05), but not in controls (
P
values >0.2). Ejection fraction and stress-induced myocardial ischemia were not associated with TWA.

Conclusions—

Mental stress can induce cardiac electrical instability, as assessed via TWA, among patients with arrhythmic vulnerability and occurs at lower heart rates than with exercise. Pathophysiological mechanisms of mental stress–induced arrhythmias may therefore involve central and autonomic nervous system pathways that differ from exercise-induced arrhythmias.
Angiotensin II Type-1 Receptor Blocker Valsartan Enhances Insulin Sensitivity in Skeletal Muscles of Diabetic Mice
Authors: Shiuchi Tetsuya , Iwai Masaru , Li Huan-Sheng , Wu Lan , Min Li-Juan , Li Jian-Mei , Okumura Midori , Cui Tai-Xing , Horiuchi Masatsugu
Published: March 2004

Angiotensin II has been shown to contribute to the pathogenesis of insulin resistance; however, the mechanism is not well understood. The present study was undertaken to investigate the potential effect of an angiotensin II type-1 (AT
1
) receptor blocker, valsartan, to improve insulin resistance and to explore the signaling basis of cross-talk of the AT
1
receptor- and insulin-mediated signaling in type 2 diabetic KK-Ay mice. Treatment of KK-Ay mice with valsartan at a dose of 1 mg/kg per day, which did not influence systolic blood pressure, significantly increased insulin-mediated 2-[
3
H]deoxy-
d
-glucose (2-[
3
H]DG) uptake into skeletal muscle and attenuated the increase in plasma glucose concentration after a glucose load and plasma concentrations of glucose and insulin. In contrast, insulin-mediated 2-[
3
H]DG uptake into skeletal muscle was not influenced in AT
2
receptor null mice, and an AT
2
receptor blocker, PD123319, did not affect 2-[
3
H]DG uptake and superoxide production in skeletal muscle of KK-Ay mice. Moreover, we observed that valsartan treatment exaggerated the insulin-induced phosphorylation of IRS-1, the association of IRS-1 with the p85 regulatory subunit of phosphoinositide 3 kinase (PI 3-K), PI 3-K activity, and translocation of GLUT4 to the plasma membrane. It also reduced tumor necrosis factor-α (TNF-α) expression and superoxide production in skeletal muscle of KK-Ay mice. Specific AT
1
receptor blockade increases insulin sensitivity and glucose uptake in skeletal muscle of KK-Ay mice via stimulating the insulin signaling cascade and consequent enhancement of GLUT4 translocation to the plasma membrane.
Adjacent Expression of Renin and Angiotensinogen in the Rostral Ventrolateral Medulla Using a Dual-Reporter Transgenic Model
Authors: Lavoie Julie L. , Cassell Martin D. , Gross Kenneth W. , Sigmund Curt D.
Published: March 2004

All components of the renin-angiotensin system are localized in the brain. However, because renin is present in very low concentrations, the mechanism by which angiotensin II is formed in the brain remains unclear. We previously reported the development of 2 transgenic mouse models using sensitive reporters, enhanced green fluorescent protein (eGFP) and β-galactosidase (β-Gal), to examine the cellular localization of renin and angiotensinogen in the mouse brain. To determine whether renin and angiotensinogen are coexpressed or present in neighboring cells in the rostral ventrolateral medulla (RVLM) and other cardiovascular control regions of the brain, we produced and examined double-transgenic mice, which express eGFP driven by the renin promoter (REN-1c/eGFP) and β-gal driven by the human angiotensinogen promoter (hAGT/β-gal). Using these reporter transgenes as sensitive markers for renin and angiotensinogen expression, we conclude that both proteins are coexpressed in the parabrachial nucleus and central nucleus of the amygdala and are in adjacent cells in the RVLM, reticular formation, bed nucleus of the stria terminalis, subfornical organ, and CA1–3 region. These data suggests that, in these areas, both renin and angiotensinogen are in close proximity providing the potential for the local formation of angiotensin I either intracellularly, when there is colocalization, or in the interstitium, when they are in juxtaposed cells.
QRS Duration and QT Interval Predict Mortality in Hypertensive Patients With Left Ventricular Hypertrophy
Authors: Oikarinen Lasse , Nieminen Markku S. , Viitasalo Matti , Toivonen Lauri , Jern Sverker , Dahlöf Björn , Devereux Richard B. , Okin Peter M.
Published: March 2004

Left ventricular hypertrophy is a risk factor for cardiovascular mortality, including sudden cardiac death. Experimentally, left ventricular hypertrophy delays ventricular conduction and prolongs action potential duration. Electrocardiographic QRS duration and QT interval measures reflect these changes, but whether these measures can further stratify risk in patients with electrocardiographic left ventricular hypertrophy is unknown. We measured the QRS duration and QT intervals from the baseline 12-lead electrocardiograms in the Losartan Intervention For Endpoint Reduction in Hypertension (LIFE) study, which included hypertensive patients with electrocardiographic evidence of left ventricular hypertrophy randomized to either losartan-based or atenolol-based treatment to lower blood pressure. In the present study, we related study baseline electrocardiographic measures to cardiovascular and all-cause mortality. There were 5429 patients (male 45.8%; mean age 66±7 years) included in the present analyses. After a mean follow-up of 4.9±0.8 years, there were 417 deaths from all causes, including 214 cardiovascular deaths. In separate univariate Cox regression analyses, QRS duration and several QT measures were significant predictors of cardiovascular mortality and all-cause mortality. However, in multivariate Cox analyses including all electrocardiographic measures and adjusting for other risk factors as well as treatment strategy, only QRS duration and maximum rate-adjusted QT
apex
interval remained as significant independent predictors of cardiovascular (
P
=0.022 and
P
=0.037, respectively) and all-cause mortality (
P
=0.038 and
P
=0.002, respectively). In conclusion, in a hypertensive risk population identified by electrocardiographic left ventricular hypertrophy, increased QRS duration and maximum QT
apex
interval can further stratify mortality risk even in the setting of effective blood pressure-lowering treatment.
Adverse Prognostic Significance of New Diabetes in Treated Hypertensive Subjects
Authors: Verdecchia Paolo , Reboldi Gianpaolo , Angeli Fabio , Borgioni Claudia , Gattobigio Roberto , Filippucci Lucia , Norgiolini Silvia , Bracco Costanza , Porcellati Carlo
Published: March 2004

Diabetes may develop in nondiabetic hypertensive subjects during treatment, but the long-term cardiovascular implications of this phenomenon are not clear. We determined the prognostic value of new diabetes in hypertensive subjects. In a long-term cohort study, 795 initially untreated hypertensive subjects, 6.5% of whom with type 2 diabetes, underwent diagnostic procedures including 24-hour ambulatory blood pressure (BP) monitoring and electrocardiography (ECG). Procedures were repeated after a median of 3.1 years in the absence of cardiovascular events. Follow-up duration was 1 to 16 years (median 6.0). New diabetes occurred in 5.8% of subjects initially without diabetes. Antihypertensive treatment included a diuretic in 53.5% of these subjects, versus 30.4% of those in whom diabetes did not develop (
P
=0.002). Plasma glucose at entry (
P
=0.0001) and diuretic treatment on follow-up (
P
=0.004) were independent predictors of new diabetes. Subsequent to the follow-up visit, a first cardiovascular event occurred in 63 subjects. Event rate in nondiabetic subjects at both visits, new diabetes, and diabetes at entry were 0.97, 3.90, and 4.70×100 person-years, respectively (
P
=0.0001). After adjustment for several confounders, including 24-hour ambulatory BP, the relative risk of events was 2.92 (95% CI: 1.33 to 6.41;
P
=0.007) in the group with new diabetes and 3.57 (95% CI: 1.65 to 7.73;
P
=0.001) in the group with previous diabetes, when compared with the group persistently free of diabetes. In treated hypertensive subjects, occurrence of new diabetes portends a risk for subsequent cardiovascular disease that is not dissimilar from that of previously known diabetes.
Extracellular Matrix Glycoprotein Biglycan Enhances Vascular Smooth Muscle Cell Proliferation and Migration
Authors: Shimizu-Hirota Ryoko , Sasamura Hiroyuki , Kuroda Mari , Kobayashi Emi , Hayashi Matsuhiko , Saruta Takao
Published: March 2004

Proteoglycans are produced and secreted by vascular smooth muscle cells, but the pathophysiological role of these glycoproteins in the vasculature is an enigma. Because the small leucine-rich proteoglycan (SLRP) biglycan is overexpressed in arteriosclerotic lesions, we produced mice constitutively overexpressing biglycan in the vascular smooth muscle, in order to examine the effects on vascular pathology. In the aorta and renal vasculature, increased vascular proliferation was seen both in the basal state and after infusion of angiotensin II (Ang II) in the transgenic mice compared with wild-type controls. In addition, the combination of biglycan overexpression and Ang II infusion resulted in marked increases in vascular smooth muscle cell proliferation and migration in the coronary arteries, as well as increases in fibrosis surrounding the vessels. In vitro, biglycan caused an increase in thymidine incorporation and migration of vascular smooth muscle cells, whereas these parameters were unchanged or reduced in endothelial cells. Moreover, addition of biglycan resulted in an increase in cdk2 expression and decrease in p27 levels in the vascular smooth muscle cells. These results suggest that this extracellular matrix SLRP may be involved in the regulation of vascular smooth muscle growth and migration through cdk2- and p27-dependent pathways. Furthermore, changes in biglycan expression could be a factor influencing the susceptibility of arteries to vascular injury, and may play a direct role in the pathogenesis of vascular lesions.
Tissue Plasminogen Activator −7351C/T Enhancer Polymorphism Is a Risk Factor for Lacunar Stroke
Authors: Jannes Jim , Hamilton-Bruce Monica A. , Pilotto Louis , Smith Brian J. , Mullighan Charles G. , Bardy Peter G. , Koblar Simon A.
Published: March 2004

Background and Purpose—

Occlusive thrombosis is an important component of small- and large-vessel ischemic stroke. Endogenous tissue plasminogen activator (TPA) is the primary mediator of intravascular fibrinolysis and is predominantly expressed by the endothelium of small vessels. The acute release of TPA is influenced by the TPA −7351C/T polymorphism and therefore may play an important role in the pathogenesis of lacunar stroke. In this study, we investigated the risk of lacunar and nonlacunar ischemic stroke associated with the TPA −7351C/T polymorphism.

Methods—

We conducted a case-control study of 182 cases of ischemic stroke and 301 community controls. Participants were evaluated for known cerebrovascular risk factors, and the TPA −7351C/T genotype was established by a polymerase chain reaction (PCR) method. Logistic regression was used to determine the risk of lacunar and nonlacunar ischemic stroke associated with the TPA −7351C/T polymorphism.

Results—

The prevalence of the TPA −7351 CC, CT, and TT genotypes were 46%, 45%, and 9% for controls and 41%, 46%, and 13% for stroke patients, respectively. After adjustment for known cerebrovascular risk factors, the TT genotype was significantly associated with ischemic stroke (OR: 1.9; 95% CI: 1.01 to 3.6). Stratification for stroke subtype showed a significant association between the TT genotype and lacunar stroke but not nonlacunar stroke (OR: 2.7; 95% CI: 1.1 to 6.7).

Conclusions—

The TPA −7351C/T polymorphism is an independent risk factor for lacunar stroke. The findings suggest that impaired fibrinolysis may play a role in the pathogenesis of lacunar stroke.