bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of packaging cell lines for generation of adeno‐associated virus vectors by lentiviral gene transfer of trans‐complementary components
Authors: Nakamura Satoki , Nakamura Reiko , Shibata Kiyoshi , Kobayashi Miki , Sahara Naohi , Shigeno Kazuyuki , Shinjo Kaori , Naito Kensuke , Ohnishi Kazunori , Kasahara Noriyuki , Iwaki Yuichi
Published: September 2004

Abstract:  Adeno‐associated virus (AAV) vector system has several useful advantages with regard to in vitro and in vivo gene transfer. However, their usages have been limited by cumbersome and labor‐intensive vector production in the traditional method. To overcome limitations in AAV production, in this report, we explored the possibility of generating AAV packaging cell line, 293T R/C.VA.E2A.E4. cells, by using lentivirus‐mediated transduction of Rep/Cap gene of AAV‐2, VA RNA, E2A, and E4 genes of Ad5 into 293T cells. In packaging cell lines, it is important that supply of the AAV vector can be stably performed for long time. We showed that the 293T R/C.VA.E2A.E4. cells have stably maintained the transduced components after more than 10 passages and yielded high‐titer AAV vectors, and the titer of AAV vectors did not decline even if culture of the packaging cells was continued for long time. The Rep/Cap and E4 gene products caused no remarkable cytotoxicity. The 293T R/C.VA.E2A.E4. cells might be able to tolerate the Rep/Cap and E4 gene products, or have less copy numbers of the Rep/Cap and E4 genes than the traditional method. Moreover, we showed that the AAV vectors derived from 293T R/C.VA.E2A.E4. cells infected the primary human CD34+ haematopoietic progenitor cells with high efficiency (50–70%). In the 293T R/C.VA.E2A.E4. cells, the AAV vectors can be generated by the transfection of one AAV vector plasmid, and large‐scale AAV production can be easily achieved. It is important that cumbersome, variable, and costly transfection is avoided.
Risk factors related to traumatic dental injuries in Brazilian schoolchildren
Authors: Soriano Evelyne Pessoa , Caldas Arnaldo França , Góes Paulo Sávio Angeiras
Published: September 2004

Abstract –  The aim of this pilot study was to analyse whether overjet, lip coverage and obesity represented risk factors associated with the occurrence of dental trauma in the permanent anterior teeth of schoolchildren in Recife, Brazil. It included a random sample of 116 boys and girls aged 12 years, attending both public and private schools. Data was collected through clinical examinations and interviews. Dental trauma was classified according to Andreasen's criteria (1994). Overjet was considered as risk factor when it presented values higher than 5 mm. Lip coverage was classified as adequate or inadequate, while obesity was considered according to National Center for Health Statistics (NCHS) procedures for the assessment of nutritional status. The prevalence of dental injuries was 23.3%. Boys experienced more injuries than girls, 30 and 16.1%, respectively (P > 0.05). There was a statistically significant difference between traumatic dental injuries and overjet (P < 0.05) and between traumatic dental injuries and lip coverage (P = 0.000). No statistical significant differences were found when obesity and dental trauma were analysed (P < 0.05). It was concluded that boys from lower social strata attending public schools, presenting an overjet size greater than 5 mm and an inadequate lip coverage, were more likely to have traumatic dental injuries in Recife, Brazil. Obesity was not a risk factor for dental trauma in this sample.
Prevalence of crown fractures in 8–10 years old schoolchildren in Canoas, Brazil
Authors: Tovo Maximiano Ferreira , Dos Santos Patricia Roth , Kramer Paulo Floriani , Feldens Carlos Alberto , Sari Gilberto Timm
Published: September 2004

Abstract –  The objective of this work was to evaluate the prevalence of children with crown fractures in permanent anterior teeth in 206 schoolchildren (104 girls and 102 boys) between the ages of 8 and 10 years, enrolled in three public schools in the city of Canoas, Brazil. The prevalence found was 17% with no significant difference between boys and girls, as well as between the ages. The most affected tooth was the maxillary central incisor, and a majority of the children showed only one affected tooth (88.6%). The types of fracture most commonly found were oblique and horizontal, and the portions of dental structures most affected were ‘enamel only’ and ‘enamel and dentin’. Only seven children (20%) sought out dental treatment.
Diagnostic characteristics of pulpal blood flow levels associated with adverse outcomes of luxated permanent maxillary incisors
Authors: Emshoff R. , Emshoff I. , Moschen I. , Strobl H.
Published: September 2004

Abstract –  Laser Doppler flowmetry (LDF) is a non‐invasive method to assess pulpal blood flow (PBF). Dental injury has been associated with significant PBF reduction. The purpose of this study was (i) to describe PBF characteristics of teeth with specific clinical outcomes, and (ii) to demonstrate diagnostic characteristics for different threshold PBF values for detection of specific multiple adverse outcomes. In 80 trauma patients, a single maxillary incisor treated by repositioning and splinting, and the respective contralateral homologous control tooth were investigated by LDF to assess local PBF values. Perfusion units (PU) were taken in two sessions, on the day of splint removal (session I), and 12 weeks after splint removal (session II). The ability of session II‐related PBF measurements at 2.9, 6.4 and 9.9 PU levels to identify adverse outcomes occurring 36 weeks after splint removal was investigated. Adverse outcomes were classified as type I (loss of sensitivity), type II (periapical radiolucency), type III (grey discolouration), type IV (loss of sensitivity and periapical radiolucency), and type V (loss of sensitivity, periapical radiolucency and grey discolouration of crown). Receiver–operator characteristic (ROC) curves were used to evaluate the sensitivity and specificity of PBF assessments. There was a significant increase in PBF values from session I to session II (P = 0.0001) for teeth without an adverse outcome, while teeth affected by a type II–V outcome showed a significant decrease in PBF values (P < 0.05). PBF measurements did not change over time for the contralateral incisors (P > 0.05). A type IV and V outcome occurred in 21 and 24% of the instances, respectively. The PBF of 2.9 PU demonstrated a sensitivity of 70% and a specificity of 93% for type V outcomes. The best likelihood ratio was found for the PBF 2.9 PU level and incisors associated with a type V outcome. The data suggest that the LDF test to be a valuable diagnostic adjunct for luxated teeth showing signs of adverse outcomes including grey discolouration or a combination of other signs. However, it may also become necessary to apply clinical decision‐making methods in order to correctly evaluate the value of information gathered. The clinical implication is that LDF may become useful in the prediction of adverse outcomes at a much earlier time period than may be accomplished by standard sensitivity tests.
Inhibition of KATP channel activity augments baroreflex‐mediated vasoconstriction in exercising human skeletal muscle
Authors: Keller David Melvin , Ogoh Shigehiko , Greene Shane , Olivencia‐Yurvati A. , Raven Peter B.
Published: September 2004

In the present investigation we examined the role of ATP‐sensitive potassium (KATP) channel activity in modulating carotid baroreflex (CBR)‐induced vasoconstriction in the vasculature of the leg. The CBR control of mean arterial pressure (MAP) and leg vascular conductance (LVC) was determined in seven subjects (25 ± 1 years, mean ±s.e.m.) using the variable‐pressure neck collar technique at rest and during one‐legged knee extension exercise. The oral ingestion of glyburide (5 mg) did not change mean arterial pressure (MAP) at rest (86 versus 89 mmHg, P > 0.05), but did appear to increase MAP during exercise (87 versus 92 mmHg, P= 0.053). However, the CBR–MAP function curves were similar at rest before and after glyburide ingestion. The CBR‐mediated decrease in LVC observed at rest (∼39%) was attenuated during exercise in the exercising leg (∼15%, P < 0.05). Oral glyburide ingestion partially restored CBR‐mediated vasoconstriction in the exercising leg (∼40% restoration, P < 0.05) compared to control exercise. These findings indicate that KATP channel activity modulates sympathetic vasoconstriction in humans and may prove to be an important mechanism by which functional sympatholysis operates in humans during exercise.
Human PAD4 Regulates Histone Arginine Methylation Levels via Demethylimination
Authors: Wang Yanming , Wysocka Joanna , Sayegh Joyce , Lee Young-Ho , Perlin Julie R. , Leonelli Lauriebeth , Sonbuchner Lakshmi S. , McDonald Charles H. , Cook Richard G. , Dou Yali , Roeder Robert G. , Clarke Steven , Stallcup Michael R. , Allis C. David , Coonrod Scott A.
Published: September 2004

Methylation of arginine (Arg) and lysine residues in histones has been correlated with epigenetic forms of gene regulation. Although histone methyltransferases are known, enzymes that demethylate histones have not been identified. Here, we demonstrate that human peptidylarginine deiminase 4 (PAD4) regulates histone Arg methylation by converting methyl-Arg to citrulline and releasing methylamine. PAD4 targets multiple sites in histones H3 and H4, including those sites methylated by coactivators CARM1 (H3 Arg
17
) and PRMT1 (H4 Arg
3
). A decrease of histone Arg methylation, with a concomitant increase of citrullination, requires PAD4 activity in human HL-60 granulocytes. Moreover, PAD4 activity is linked with the transcriptional regulation of estrogen-responsive genes in MCF-7 cells. These data suggest that PAD4 mediates gene expression by regulating Arg methylation and citrullination in histones.
Observation of Superflow in Solid Helium
Authors: Kim E. , Chan M. H. W.
Published: September 2004

We report on the observation of nonclassical rotational inertia in solid helium-4 confined to an annular channel in a sample cell under torsional motion, demonstrating superfluid behavior. The effect shows up as a drop in the resonant oscillation period as the sample cell is cooled below 230 millikelvin. Measurement of 17 solid samples allows us to map out the boundary of this superfluid-like solid or supersolid phase from the melting line up to 66 bars. This experiment indicates that superfluid behavior is found in all three phases of matter.
Avian H5N1 Influenza in Cats
Authors: Kuiken Thijs , Rimmelzwaan Guus , van Riel Debby , van Amerongen Geert , Baars Marianne , Fouchier Ron , Osterhaus Albert
Published: September 2004

During the 2003 to 2004 outbreak of avian influenza A (H5N1) virus in Asia, there were anecdotal reports of fatal infection in domestic cats, although this species is considered resistant to influenza. We experimentally inoculated cats with H5N1 virus intratracheally and by feeding them virus-infected chickens. The cats excreted virus, developed severe diffuse alveolar damage, and transmitted virus to sentinel cats. These results show that domestic cats are at risk of disease or death from H5N1 virus, can be infected by horizontal transmission, and may play a role in the epidemiology of this virus.
Temporal relation between depression and cognitive impairment in old age: prospective population based study
Authors: Vinkers David J , Gussekloo Jacobijn , Stek Max L , Westendorp Rudi G J , Mast Roos C van der
Published: September 2004

Objective
To examine the temporal relation between depression and cognitive impairment in old age.

Design
Prospective, population based study with four years of follow up.

Setting
City of Leiden, the Netherlands.

Participants
500 people aged 85 years at recruitment.

Main outcome measures
Annual assessments of depressive symptoms (15 item geriatric depression scale), global cognitive function (mini-mental state examination), attention (Stroop test), processing speed (letter digit coding test), and immediate and delayed recall (12 word learning test).

Results
At 85 years old, participants' depressive symptoms and cognitive impairment were highly significantly correlated (P < 0.001). During follow up, an accelerated annual increase of depressive symptoms was associated with impaired attention (0.08 points (95% confidence interval 0.01 to 0.16)), immediate recall (0.17 points (0.09 to 0.25)), and delayed recall (0.10 points (0.02 to 0.18)) at baseline. In contrast, depressive symptoms at baseline were not related to an accelerated cognitive decline during follow up (P > 0.05).

Conclusion
Caregivers should be aware of the development of depressive symptoms when cognitive impairment is present. However, the presence of depression only does not increase the risk of cognitive decline.
Inhibition of ACh-stimulated exocytosis by NSAIDs in guinea pig antral mucous cells: autocrine regulation of mucin secretion by PGE2
Authors: Shimamoto Chikao , Fujiwara Shoko , Kato Masumi , Ito Shigenori , Katsu Ken-ichi , Mori Hiroshi , Nakahari Takashi
Published: September 2004

The effects of indomethacin (IDM) and aspirin (ASA) on ACh (10 μM) -stimulated exocytotic events were studied in guinea pig antral mucous cells by using video optical microscopy. IDM or ASA, which inhibits cyclooxygenase (COX), decreased the frequency of ACh-stimulated exocytotic events by 30% or 60%, respectively. The extent of inhibition induced by ASA (60%) decreased by 30% when IDM or arachidonic acid (AA, the substrate of COX) was added. IDM, unlike ASA, appears to induce the accumulation of AA, which enhances the frequency of ACh-stimulated exocytotic events in ASA-treated cells. ONO-8713 (100 μM; an inhibitor of the EP1–EP4 prostaglandin receptors) and N-[2-(( p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, HCl (H-89, 20 μM; an inhibitor of PKA) also decreased the frequency of ACh-stimulated exocytotic events by 60%. However, the supplementation of PGE2 (1 μM) prevented the IDM-induced decrease in the frequency of ACh-stimulated exocytotic events. SC-560 (an inhibitor of COX-1) decreased the frequency of ACh-stimulated exocytotic events by 30%, but NS-398 (an inhibitor of COX-2) did not. Moreover, IDM decreased the frequency of exocytotic events stimulated by ionomycin, suggesting that COX-1 activity is stimulated by an increase in intracellular Ca2+ concentration ([Ca2+]i). ACh and ionomycin increased PGE2 release in antral mucosal cells. In conclusion, in ACh-stimulated antral mucous cells, an increase in [Ca2+]i activates Ca2+-regulated exocytotic events and PGE2 release mediated by COX-1. The released PGE2 induces the accumulation of cAMP, which enhances the Ca2+-regulated exocytosis. The autocrine mechanism mediated by PGE2 maintains the high-level mucin release from antral mucous cells during ACh stimulation.
Direct observation of epicardial coronary capillary hemodynamics during reactive hyperemia and during adenosine administration by intravital video microscopy
Authors: Kiyooka Takahiko , Hiramatsu Osamu , Shigeto Fumiyuki , Nakamoto Hiroshi , Tachibana Hiroyuki , Yada Toyotaka , Ogasawara Yasuo , Kajiya Masahito , Morimoto Taro , Morizane Yuki , Mohri Satoshi , Shimizu Juichiro , Ohe Tohru , Kajiya Fumihiko
Published: September 2004

Using high-resolution intravital charge-coupled device video microscopy, we visualized the epicardial capillary network of the beating canine heart in vivo to elucidate its functional role under control conditions, during reactive hyperemia (RH), and during intracoronary adenosine administration. The pencil-lens video-microscope probe was placed over capillaries fed by the left anterior descending artery in atrioventricular-blocked hearts of open-chest, anesthetized dogs paced at 60–90 beats/min ( n = 17). In individual capillaries under control conditions, red blood cell flow was predominant during systole or diastole, indicating that the watershed between diastolic arterial and systolic venous flows is located within the capillaries. Capillary flow increased during RH and reached a peak flow velocity (2.1 ± 0.6 mm/s), twice as high as control (1.2 ± 0.5 mm/s), with enhancement of intercapillary cross-connection flow and enlargement of diameter (by 17%). With adenosine, capillary flow velocity significantly increased (1.8 ± 0.7 mm/s). However, the increase in volumetric capillary flow with adenosine estimated from red blood cell velocity and diameter was less than the increase in arterial flow, whereas that during RH was nearly equivalent to the increase in arterial flow. There was a time lag of ∼1.5 s for refilling of capillaries during RH, indicating their function as capacitance vessels. In conclusion, the coronary capillary network functions as 1) the major watershed between diastolic-dominant arterial and systolic-dominant venous flows, 2) a capacitor, and 3) a significant local flow amplifier and homogenizer of blood supply during RH, but with adenosine the increase in capillary flow velocity was less than the increase in arterial flow.
Why is flow-mediated dilation dependent on arterial size? Assessment of the shear stimulus using phase-contrast magnetic resonance imaging
Authors: Silber Harry A. , Ouyang Pamela , Bluemke David A. , Gupta Sandeep N. , Foo Thomas K. , Lima Joao A. C.
Published: September 2004

Flow-mediated dilation (FMD) is strongly dependent on arterial size, but the reasons for this phenomenon are poorly understood. We have previously shown that FMD is greater in small brachial arteries because the shear stress stimulus is greater in small brachial arteries. However, it is unclear why the shear stimulus is greater in small arteries. Furthermore, this relationship has not been investigated in other, differently sized arterial beds. Postischemic systolic shear stress and resulting FMD were evaluated in the brachial and femoral arteries of 24 young, healthy adults using phase-contrast magnetic resonance imaging. Arterial shear and radius were calculated from the velocity profile via a best-fit parabola before and after occlusion. Summing the velocity pixels provided hyperemic systolic flow. FMD was proportional to hyperemic shear in the brachial and femoral arteries ( P < 0.0001, r = 0.60). Hyperemic systolic flow was proportional to radius2 ( P < 0.0001, r = 0.93). Applying this relationship to the Poiseuille equation (shear is proportional to flow/radius3) shows that hyperemic shear is proportional to radius2/radius3 and, therefore, explains why hyperemic shear is proportional to 1/radius. We conclude that FMD is proportional to hyperemic systolic shear stress in both the brachial and the femoral arteries. The hyperemic shear stimulus for FMD is greater in small arteries due to the dependence of postischemic systolic flow on radius squared. Therefore, greater FMD in small arteries does not necessarily reflect better conduit artery endothelial function. Evaluating the shear stimulus using phase-contrast magnetic resonance imaging enhances the understanding of mechanisms underlying FMD.
Effects of PPAR-γ ligands on vascular smooth muscle marker expression in hypertensive and normal arteries
Authors: Atkins Kevin B. , Northcott Carrie A. , Watts Stephanie W. , Brosius Frank C.
Published: September 2004

Having previously demonstrated that glucose transporter-4 (GLUT4) expression was reduced in aortas and carotid arteries of deoxycorticosterone acetate (DOCA) salt-hypertensive rats, we hypothesized that troglitazone (TG), through activation of peroxisome proliferator-activated receptor-γ (PPAR-γ), would stabilize GLUT4 expression and possibly preserve the differentiated phenotype in vascular smooth muscle cells. In DOCA salt-hypertensive rats treated with TG (100 mg/day), there was a significant ( P < 0.001) decrease in systolic blood pressure (BP; 149.9 ± 4.4 mmHg) compared with the untreated DOCA salt-hypertensive rats (202.2 ± 10.34 mmHg). Separate trials with rosiglitazone (RS; 3 mg/day) demonstrated a significant ( P < 0.001) decrease in BP (DOCA salt, 164.2 ± 9.8 vs. DOCA-RS, 124.9 ± 3.7 mmHg) comparable to that with TG. Expression of GLUT4, h-caldesmon, and smooth muscle myosin heavy chain SM2 was significantly decreased in aortas of DOCA salt-hypertensive rats and was reversed by TG to levels similar to those in aortas of sham-treated rats. TG (50 μM) induced GLUT4 and h-caldesmon expression in 24-h culture of explanted carotid arteries of DOCA salt-hypertensive rats, and the endogenous PPAR-γ ligand 15-deoxy-Δ12–14-prostaglandin J2 (PGJ2; 20 μM) and TG (50 μM) similarly increased GLUT4, h-caldesmon, and SM2 protein expression in explanted aortas. The expression of activated, phosphorylated Akt was increased by PGJ2 and TG with no significant effect on total Akt levels. Inhibition of phosphorylated Akt expression using the phosphatidylinositol 3-kinase inhibitor LY-294002 (16 μM) abrogated the increased expression of h-caldesmon and SM2. These data demonstrate that PPAR-γ agonists maintain or induce expression of markers of the contractile phenotype independently of their effects on hypertension, and that this effect may be mediated through activation of phosphatidylinositol 3-kinase/Akt.
Effects of menstrual cycle and oral contraceptive use on calf venous compliance
Authors: Meendering Jessica R. , Torgrimson Britta N. , Houghton Belinda L. , Halliwill John R. , Minson Christopher T.
Published: September 2004

Numerous studies have shown that the female sex hormones estrogen and progesterone have multiple effects on the vasculature. Thus our goal was to investigate the effects of estrogen and progesterone on calf venous compliance by looking for cyclic changes during the early follicular, ovulatory, and midluteal phases of the menstrual cycle and during high and low hormone phases of oral contraceptive use. Additionally, we wanted to compare the venous compliance of normally menstruating women, oral contraceptive users, and men. We studied eight normally menstruating women (23 ± 1 yr of age) during the early follicular, ovulatory, and midluteal phases of the menstrual cycle. Nine triphasic oral contraceptive users (21 ± 1 yr of age) were studied during weeks of high and low hormone concentrations. Eight men (23 ± 1 yr of age) were studied twice within 2–4 wk. With the use of venous occlusion plethysmography with mercury in-Silastic strain gauges, lower limb venous compliance was measured by inflating a venous collection cuff that was placed on the thigh to 60 mmHg for 8 min and then reducing the pressure to 0 mmHg at a rate of 1 mmHg/s. Venous compliance was calculated as the derivative of the pressure-volume curves. There were no differences between early follicular, ovulatory, and midluteal phases of the menstrual cycle or between high and low hormone phases of oral contraceptive use ( P > 0.05). Male venous compliance was significantly greater than in normally menstruating women ( P < 0.001) and oral contraceptive users ( P < 0.002). These data support a sex difference but also suggest that venous compliance does not change with menstrual cycle phase or during the course of oral contraceptive use.
Impact of combined NO and PG blockade on rapid vasodilation in a forearm mild-to-moderate exercise transition in humans
Authors: Saunders Natasha R. , Dinenno Frank A. , Pyke Kyra E. , Rogers Anna M. , Tschakovsky Michael E.
Published: September 2004

We tested the hypothesis that nitric oxide (NO) and prostaglandins (PGs) contribute to the rapid vasodilation that accompanies a transition from mild to moderate exercise. Nine healthy volunteers (2 women and 7 men) lay supine with forearm at heart level. Subjects were instrumented for continuous brachial artery infusion of saline (control condition) or combined infusion of NG-nitro-l-arginine methyl ester (l-NAME) and ketorolac (drug condition) to inhibit NO synthase and cyclooxygenase, respectively. A step increase from 5 min of steady-state mild (5.4 kg) rhythmic, dynamic forearm handgrip exercise (1 s of contraction followed by 2 s of relaxation) to moderate (10.9 kg) exercise for 30 s was performed. Steady-state forearm blood flow (FBF; Doppler ultrasound) and forearm vascular conductance (FVC) were attenuated in drug compared with saline (control) treatment: FBF = 196.8 ± 30.8 vs. 281.4 ± 34.3 ml/min and FVC = 179.3 ± 29.4 vs. 277.8 ± 34.8 ml·min−1·100 mmHg−1 (both P < 0.01). FBF and FVC increased from steady state after release of the initial contraction at the higher workload in saline and drug conditions: ΔFBF = 72.4 ± 8.7 and 52.9 ± 7.8 ml/min, respectively, and ΔFVC = 66.3 ± 7.3 and 44.1 ± 7.0 ml·min−1·100 mmHg−1, respectively (all P < 0.05). The percent ΔFBF and ΔFVC were not different during saline infusion or combined inhibition of NO and PGs: ΔFBF = 27.2 ± 3.1 and 28.1 ± 3.8%, respectively ( P = 0.78) and ΔFVC = 25.7 ± 3.2 and 26.0 ± 4.0%, respectively ( P = 0.94). The data suggest that NO and vasodilatory PGs are not obligatory for rapid vasodilation at the onset of a step increase from mild- to moderate-intensity forearm exercise. Additional vasodilatory mechanisms not dependent on NO and PG release contribute to the immediate and early increase in blood flow in an exercise-to-exercise transition.
Actions of neuropeptide W in paraventricular hypothalamus: implications for the control of stress hormone secretion
Authors: Taylor Meghan M. , Yuill Erik A. , Baker Jennifer R. , Ferri Catharine C. , Ferguson Alastair V. , Samson Willis K.
Published: September 2004

Neuropeptide W (NPW) is produced in neurons located in hypothalamus and brain stem, and its receptors are present in the hypothalamus, in particular in the paraventricular nucleus (PVN). Intracerebroventricular (ICV) administration of NPW activated, in a dose-related fashion, the hypothalamic-pituitary-adrenal axis, as determined by plasma corticosterone levels in conscious rats but, at those same doses, did not stimulate the release of oxytocin or vasopressin into the peripheral circulation or alter blood pressure or heart rate. The ability of ICV-administered NPW to stimulate the hypothalamic-pituitary-adrenal axis in conscious male rats was blocked by intravenous pretreatment with a corticotropin-releasing hormone antagonist. This suggested an action of NPW in the parvocellular division of the PVN. Indeed, in hypothalamic slice preparations (whole cell patch recording), bath application of NPW depolarized and increased the spike frequency of the majority of electrophysiologically identified putative neuroendocrine PVN neurons. Effects on membrane potential were maintained in the presence of TTX, suggesting them to be direct postsynaptic actions on these neuroendocrine cells. Our data suggest that endogenous NPW, produced in brain, may play a physiologically relevant role in the neuroendocrine response to stress.
Asynchronous Shear Stress and Circumferential Strain Reduces Endothelial NO Synthase and Cyclooxygenase-2 but Induces Endothelin-1 Gene Expression in Endothelial Cells
Authors: Dancu Michael B. , Berardi Danielle E. , Vanden Heuvel John P. , Tarbell John M.
Published: September 2004

Objective—Endothelium-derived vasoactive agents NO, endothelin-1 (ET-1), and prostacyclin (PGI2) not only regulate vascular tone but also influence atherogenic processes, including smooth muscle migration and proliferation, as well as monocyte and platelet adhesion. Complex hemodynamics characterized by the temporal phase angle between mechanical factors circumferential strain and wall shear stress (stress phase angle [SPA]) have been implicated in regions prone to pathologic development, such as atherosclerosis and intimal hyperplasia, in coronary and peripheral arteries where the mechanical forces are highly asynchronous (SPA=−180°). We determined the gene expression of endothelial NO synthase (eNOS), ET-1, and cyclooxygenase-2 (COX-2) affected by asynchronous hemodynamics (SPA=−180°) relative to normal hemodynamics (SPA=0°) in bovine aortic endothelial cells.

Methods and Results—Quantitative competitive RT-PCR analysis showed that eNOS production (at 5 and 12 hours) and COX-2 production (at 5 hours) were reduced at the gene expression level by asynchronous hemodynamics (SPA=−180°) compared with synchronous hemodynamics (SPA=0°), whereas ET-1 exhibited an opposite trend (at 5 and 12 hours). NO, ET-1, and PGI2secretion followed their respective gene expression profiles after 5 and 12 hours.

Conclusion—Together, these data suggest that highly asynchronous mechanical force patterns (SPA=−180°) can elicit proatherogenic vasoactive responses in endothelial cells at the gene expression level, indicating a novel mechanism that induces cardiovascular pathology.
Genetic Influence on Inflammation Variables in the Elderly
Authors: de Maat Moniek P.M. , Bladbjerg Else Marie , von Bornemann Hjelmborg Jacob , Bathum Lise , Jespersen Jørgen , Christensen Kaare
Published: September 2004

Background—Inflammation variables (C-reactive protein [CRP], fibrinogen, and soluble intercellular adhesion molecule-1 [sICAM-1]) have been identified as risk factors for cardiovascular disease. It is still not known how much the regulation of inflammatory risk factors is determined by genetic factors, and the aim of this study was to determine the heritability of these inflammation variables and of the acute phase regulating cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) at older ages.

Methods and Results—The heritability of CRP, fibrinogen, sICAM-1, IL-6, and TNF-α was determined in a twin study consisting of 129 monozygotic twin pairs and 153 dizygotic same-sex twins aged 73 to 94 years who participated in the Longitudinal Study of Aging of Danish Twins. Furthermore, we determined the influence of selected genetic polymorphisms on the plasma level variations. Genetic factors accounted for 20% to 55% of the variation in plasma levels of the inflammation variables. The highest heritability was found for sICAM-1. The genetic polymorphisms we studied explained only a small, insignificant part of the heritability.

Conclusions—This study in elderly twins provides evidence for a substantial genetic component of inflammatory cardiovascular risk factors among the elderly.
β3-Integrin Regulates Vascular Endothelial Growth Factor-A–Dependent Permeability
Authors: Robinson Stephen D. , Reynolds Louise E. , Wyder Lorenza , Hicklin Daniel J. , Hodivala-Dilke Kairbaan M.
Published: September 2004

Objective—

β3-integrin deficiency has been implicated in increasing levels of Flk-1 expression on endothelial cells and enhancing vascular endothelial growth factor (VEGF)-induced angiogenesis. We determined the role of β3-integrin in mediating VEGF-A–induced blood vessel permeability through Flk-1.

Methods and Results—

Using the Miles assay, we demonstrated that VEGF-A–induced plasma leakage was enhanced in β3-null mice when compared with wild-type controls. This was not caused by any changes in blood vessel structure (as detected by light or electron microscopy) or by changes in endothelial cell–cell adhesion proteins (as determined by Western blot analysis, flow cytometry, and immunofluorescence). Circulating levels of VEGF, baseline blood vessel leakage, and leakage in response to an acute inflammatory stimulus were identical in wild-type and β3-null mice. However, VEGF-A–induced leakage was abolished in β3-null mice by the inhibition of Flk-1, indicating that the elevated levels of Flk-1 on β3-null endothelial cells enhance VEGF-A–induced permeability.

Conclusions—

β3-integrin–deficiency increases the sensitivity of endothelial cells to VEGF-A by elevating Flk-1 expression and, as a consequence, enhances VEGF-A–mediated permeability.
Id Family of Transcription Factors and Vascular Lesion Formation
Authors: Forrest Scott , McNamara Coleen
Published: September 2004

Vascular smooth muscle cell (VSMC) modulation to a de-differentiated phenotype and proliferation are key components of vascular lesion formation. Understanding how these processes are regulated is essential to understanding the progression of vascular diseases such as atherosclerosis and in-stent restenosis. The Id family of helix-loop-helix (HLH) transcription factors has emerged as important regulators of cellular growth and differentiation. Recent published findings have implicated the Id proteins as important regulators of growth and phenotypic modulation in VSMC and in the vascular response to injury. In this review, we summarize what is known regarding how the Id proteins function to control cellular growth and differentiation and their role in vascular lesion formation.
Inhibition of Tumor Necrosis Factor-α Reduces Atherosclerosis in Apolipoprotein E Knockout Mice
Authors: Brånén Lena , Hovgaard Lars , Nitulescu Mihaela , Bengtsson Eva , Nilsson Jan , Jovinge Stefan
Published: September 2004

Objective—Inflammation plays an important role in atherosclerosis. One of the most potent pro-inflammatory cytokines is tumor necrosis factor-α (TNF-α), a cytokine identified to have a pathogenic role in chronic inflammatory diseases such as rheumatoid arthritis (RA). The aim of the study was to evaluate the importance of TNF-α in atherogenesis.

Methods and Results—Mice deficient in both apolipoprotein E (apoE) and TNF-α were compared regarding their atherosclerotic burden. Mice were fed a Western-style diet (WD) or normal chow. Mice deficient in both apoE and TNF-α exhibited a 50% (P=0.035) reduction of relative lesion size after 10 weeks of WD. Bone marrow transplantation ofapoEomice withapoEotnf-αobone marrow resulted in a 83% (P=0.021) reduction after 25 weeks on WD. InapoEknockout mice treated with recombinant soluble TNF receptor I releasing pellets, there was a reduction in relative lesion size after 25 weeks of 75% (P=0.018).

Conclusions—These findings demonstrate that TNF-α is actively involved in the progression of atherosclerosis. Accordingly, TNF-α represents a possible target for prevention of atherosclerosis. This may be of particular importance in rheumatoid arthritis because these patients have an increased risk for cardiovascular disease.
Transcription Factor CHF1/Hey2 Regulates Neointimal Formation In Vivo and Vascular Smooth Muscle Proliferation and Migration In Vitro
Authors: Sakata Yasuhiko , Xiang Fan , Chen Zhiping , Kiriyama Yoriko , Kamei Caramai N. , Simon Daniel I. , Chin Michael T.
Published: September 2004

Objective—

To determine the role of the cardiovascular-restricted, hairy-related bHLH transcription factor, CHF1/Hey2, in the biological response to vascular injury.

Methods and Results—

We investigated the response of CHF1/Hey2-deficient mice to vascular injury in vivo and the response of primary cultured vascular smooth muscle cells (VSMCs) from these mice to growth factors in vitro. Neointima formation after arterial wire injury is decreased in knockout (KO) compared with wild-type (WT) mice (0.025±0.011 mm
2
in WT [n=13]) versus 0.016±0.008 mm
2
in KO (n=12;
P
<0.05) and is accompanied by reduced cellular proliferation. CHF1/Hey2-deficient VSMCs proliferate slowly compared with WT VSMCs and also show decreased migration in response to platelet-derived growth factor (PDGF) (62.6±10.3 CPF versus 37.2±13.5 CPF;
P
<0.01) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) (27.4±7.7 CPF versus 6.4±3.7 CPF,
P
<0.05). Furthermore, lamellipodia formation and membrane ruffling induced by these chemoattractants are diminished in KO VSMCs, which is correlated with decreased activation of the small GTPase Rac1. Although total Rac1 protein was not changed in KO VSMCs, the level of the Rac guanine exchange factor (GEF), Sos1, was decreased.

Conclusions—

CHF1/Hey2 is an important regulator of vascular smooth muscle cell (VSMC) accumulation during vascular remodeling and responsiveness to growth factors in vitro.
Lipoprotein(a)
Authors: Berglund Lars , Ramakrishnan Rajasekhar
Published: September 2004

Lipoprotein (a) [Lp(a)], is present only in humans, Old World nonhuman primates, and the European hedgehog. Lp(a) has many properties in common with low-density lipoprotein (LDL) but contains a unique protein, apo(a), which is structurally different from other apolipoproteins. The size of the apo(a) gene is highly variable, resulting in the protein molecular weight ranging from 300 to 800 kDa; this large variation may be caused by neutral evolution in the absence of any selection advantage. Apo(a) influences to a major extent metabolic and physicochemical properties of Lp(a), and the size polymorphism of the apo(a) gene contributes to the pronounced heterogeneity of Lp(a). There is an inverse relationship between apo(a) size and Lp(a) levels; however, this pattern is complex. For a given apo(a) size, there is a considerable variation in Lp(a) levels across individuals, underscoring the importance to assess allele-specific Lp(a) levels. Further, Lp(a) levels differ between populations, and blacks have generally higher levels than Asians and whites, adjusting for apo(a) sizes. In addition to the apo(a) size polymorphism, an upstream pentanucleotide repeat (TTTTAn) affects Lp(a) levels. Several meta-analyses have provided support for an association between Lp(a) and coronary artery disease, and the levels of Lp(a) carried in particles with smaller size apo(a) isoforms are associated with cardiovascular disease or with preclinical vascular changes. Further, there is an interaction between Lp(a) and other risk factors for cardiovascular disease. The physiological role of Lp(a) is unknown, although a majority of studies implicate Lp(a) as a risk factor.
Mechanisms Underlying Conduction Slowing and Arrhythmogenesis in Nonischemic Dilated Cardiomyopathy
Authors: Akar Fadi G. , Spragg David D. , Tunin Richard S. , Kass David A. , Tomaselli Gordon F.
Published: September 2004

Heart Failure (HF) is associated with an increased risk of sudden death caused by ventricular tachyarrhythmias. Recent studies have implicated repolarization abnormalities and, in particular, exaggerated heterogeneity of transmural repolarization in the genesis of polymorphic ventricular tachycardia in a canine model of nonischemic dilated cardiomyopathy. The presence and degree to which conduction abnormalities play a role in arrhythmogenesis in this model are uncertain. HF was produced in dogs by rapid RV-pacing for 3 to 4 weeks. High-resolution optical action potentials were recorded from epicardial and endocardial surfaces of arterially perfused canine wedge preparations isolated from LV and RV of normal and failing dogs. Cellular and molecular determinants of conduction were investigated using patch-clamp recordings, Western blot analysis, and immunocytochemistry. HF was associated with marked prolongation (by 33%) of the QRS duration of the volume conducted electrocardiogram and significant (>20%) slowing of epicardial and endocardial conduction velocities (CV) in both LV and RV. Cx43 expression was reduced by >40% in epicardial and endocardial layers of the LV, but was unchanged in the RV of failing hearts. Despite greater epicardial than endocardial Cx43 expression, epicardial CV was consistently slower (
P
<0.01). Immunocytochemical analysis revealed predominant colocalization of Cx43 with N-cadherin in normal versus failing samples, because Cx43 was redistributed from the intercalated disk to lateral cell borders in failing tissue. Moreover, a significant (
P
<0.05) increase in hypophosphorylated Cx43 was detected in the LV and RV of failing hearts. Action potential upstroke velocities in isolated ventricular myocytes from normal and failing hearts were not different (
P
=0.8, not significant), and Masson trichrome staining revealed no significant change in fibrosis content in HF. Nonischemic dilated cardiomyopathy is associated with significant slowing of CV that was not directly related to reduced Cx43 expression. Changes in phosphorylation and localization of Cx43 may contribute to gap-junction dysfunction, CV slowing, and arrhythmias in HF.
Central Nervous System Entry of Peripherally Injected Umbilical Cord Blood Cells Is Not Required for Neuroprotection in Stroke
Authors: Borlongan Cesar V. , Hadman Martin , Davis Sanberg Cyndy , Sanberg Paul R.
Published: September 2004

Background and Purpose—

To date, stem cell graft-mediated neuroprotection is equated with graft survival and secretion of neurotrophic factors in the brain. Here, we examined whether neuroprotection by systemically delivered human umbilical cord blood (HUCB) cells was dependent on their entry into the central nervous system in a rodent model of acute stroke.

Methods—

Adult male Sprague-Dawley rats were subjected to right middle cerebral artery occlusion for 60 minutes. During the 1-hour occlusion, animals were randomly assigned to 1 of the following treatments: intravenous injection of HUCB (a subtherapeutic dose of 200 000 cells in 10 μL) with blood–brain barrier (BBB) permeabilizer (1.1 mol/L mannitol at 4°C) or vehicle, intravenous vehicle alone, or intravenous mannitol alone. Behavioral tests, using elevated body swing test and passive avoidance test, were conducted at day 3 poststroke, and thereafter, animals were euthanized for: (1) immunohistochemical examination of HUCB, which were lentivirally labeled with green fluorescent protein; (2) cerebral infarction analysis using 2,3,5-triphenyl-tetrazolium chloride; and (3) enzyme-linked immunosorbent assay of trophic factors within the striatal region.

Results—

We did not detect intravenously administered low dose of HUCB cells in the brains of animals at day 3 after stroke even when cells were coinfused with a BBB permeabilizer (mannitol). However, HUCB–mannitol treatment significantly increased brain levels of neurotrophic factors, which correlated positively with reduced cerebral infarcts and improved behavioral functions.

Conclusions—

Our data show that central nervous system availability of grafted cells is not a prerequisite for acute neuroprotection provided that therapeutic molecules secreted by these cells could cross the BBB.
Transplantation of Circulating Endothelial Progenitor Cells Restores Endothelial Function of Denuded Rabbit Carotid Arteries
Authors: He Tongrong , Smith Leslie A. , Harrington Sean , Nath Karl A. , Caplice Noel M. , Katusic Zvonimir S.
Published: September 2004

Background and Purpose—

Circulating endothelial progenitor cells (EPCs) play an important role in repair of injured vascular endothelium and neovascularization. The present study was designed to determine the effect of EPCs transplantation on the regeneration of endothelium and recovery of endothelial function in denuded carotid arteries.

Methods—

Isolated mononuclear cells from rabbit peripheral blood were cultured in endothelial growth medium for 7 days, yielding EPCs. A rabbit model of common carotid artery denudation by passage of a deflated balloon catheter was used to evaluate the effects of EPCs on endothelial regeneration and vasomotor function. Immediately after denudation, autologous EPCs (10
5
cells in 200 μL saline) or 200 μL saline alone (control) were administered into the lumen of injured artery.

Results—

Four weeks after transplantation, fluorescence-labeled colonies of EPCs were found in the vessel wall. Local transplantation of EPCs as compared with saline administration accelerated endothelialization and significantly improved endothelium-dependent relaxation when assessed 4 weeks after denudation (n=4 to 5,
P
<0.05). Transplantation of EPCs did not affect vasomotor function of arterial smooth muscle cells. Protein array analysis of conditioned media obtained from cultured EPCs demonstrated the ability of these cells to produce and release a number of proangiogenic cytokines.

Conclusions—

We conclude that local delivery of cultured circulating EPCs into the lumen of denuded carotid arteries accelerates endothelialization and improves endothelial function. Paracrine effects of EPCs may contribute to regenerative properties of EPCs.
Complications and Pitfalls in Rat Stroke Models for Middle Cerebral Artery Occlusion
Authors: Gerriets Tibo , Stolz Erwin , Walberer Maureen , Müller Clemens , Rottger Carina , Kluge Alexander , Kaps Manfred , Fisher Marc , Bachmann Georg
Published: September 2004

Background and Purpose—

Investigating focal cerebral ischemia requires animal models that are relevant to human stroke. Complications and side effects are common among these models. The present study describes potential pitfalls in 3 techniques for middle cerebral artery occlusion (MCAO) in rats using magnetic resonance imaging (MRI) and magnetic resonance angiography (MRA).

Methods—

Rats were subjected to temporary MCAO for 90 minutes using the suture technique (group I; n=10) or to permanent MCAO using the suture technique (group II; n=10) or the macrosphere technique (group III; n=10). Clinical evaluation was performed after 3 hours and 24 hours. After 24 hours, animals underwent MRI and MRA to determine lesion size and the intracranial vascular status.

Results—

Hemispheric lesion volume was significantly smaller in group I (14.6%) compared with groups II (35.2%;
P
<0.01) and III (21.3%;
P
<0.05). Two animals (1 each in group II and III) did not demonstrate neurological deficits and had no lesion on MRI and a patent MCA main stem on MRA. Subarachnoid hemorrhage was detected in 2 animals (1 each in group I and II). MRA indicated a patent MCA main stem in 2 animals (group II), although both rats displayed neurological deficits. Hypothalamic infarction with subsequent pathological hyperthermia was detected in all animals in group II and in 1 rat in group III.

Conclusions—

Model failures occurred frequently in all groups. MRI and MRA helps to identify animals that need to be excluded from experimental stroke studies.
Reorganizing the System of Care Surrounding Laparoscopic Surgery: A Cost-Effectiveness Analysis Using Discrete-Event Simulation
Authors: Stahl James E. , Rattner David , Wiklund Richard , Lester Jessica , Beinfeld Molly , Gazelle G. Scott
Published: September 2004

Purpose. To determine the cost-effectiveness of a proposed reorganization of surgical and anesthesia care to balance patient volume and safety. Methods. Discrete-event simulation methods were used to compare current surgical practice with a newmodular system in which patient care is handed off between 2 anesthesiologists. Ahealth care system’s perspective, using hospital and professional costs, was chosen for the cost-effectiveness analysis. Outcomes were patient throughput, flow time, wait time, and resource use. Sensitivity analyses were performed on staffing levels, mortality rates, process times, and scheduled patient volume. Results. The new strategy was more effective (average 4.41 patients/d [median = 5] v. 4.29 [median = 4]) and had similar costs (average cost/ patient/d = $5327 v. $5289) to the current strategywith an incremental cost-effectiveness of $318/additional patient treated/d. Surgical mortality rate must be >4% or hand-off delay >15min before the new strategy is no longermore effective. Conclusion.The proposed system is more cost-effective relative to current practice over a wide range of mortality rates, hand-off times, and scheduled patient volumes.
The Cost-Effectiveness of Screening Programs Using Single and Multiple Birth Cohort Simulations: A Comparison Using a Model of Cervical Cancer
Authors: Dewilde Sarah , Anderson Rob
Published: September 2004

Despite early recognition of the theoretical advantages of simulations that include different population subgroups/ strata and different birth cohorts, manymodeling-based economic evaluations of cervical screening have been based on unrealistic single birth cohort simulations. The authors examined the effect of amultiple birth cohort simulation on the incremental cost-effectiveness estimates of cervical screening programs, compared to a conventional single cohort simulation. The choice of hypothetical cohort that starts the simulation had a major impact on the cost-effectiveness estimates: Compared with a single birth cohort simulation, the incremental cost-effectiveness of a shift from biennial to triennial screening was 30% higher when using the multiple cohort simulation. Multiple cohort simulations using the different age structures of 4 countries had little impact on the costeffectiveness ratios (variation <5%). Future modeling-based evaluations of screening policies should better reflect the age range of the population that is targeted by carefully specifying the nature of the starting cohort(s).