bluuebunny/crossref_metadata_2025 huggingface dataset:
Positron-Emission Tomography Reporter Gene Expression Imaging in Rat Myocardium
Authors: Inubushi Masayuki , Wu Joseph C. , Gambhir Sanjiv S. , Sundaresan Gobalakrishnan , Satyamurthy Nagichettiar , Namavari Mohammad , Yee Simon , Barrio Jorge R. , Stout David , Chatziioannou Arion F. , Wu Lily , Schelbert Heinrich R.
Published: January 2003

Background—

This study examines the quantitative accuracy, detection sensitivity, and time course of imaging the expression of a mutant herpes simplex type-1 virus thymidine kinase (HSV1-sr39tk) PET reporter gene in rat myocardium by using the PET reporter probe 9-(4-[
18
F]-Fluoro-3-Hydroxymethylbutyl)-Guanine ([
18
F]-FHBG) and a small-animal PET (microPET).

Methods and Results—

In 40 rats, adenovirus expressing HSV1-sr39tk driven by a cytomegalovirus promoter (Ad-CMV-HSV1-sr39tk, 1×10
6
to 1×10
9
pfu) was injected through a thoracotomy directly into the left ventricular myocardium. After 3 days, myocardial perfusion was imaged with [
13
N]-ammonia for delineating the left ventricular myocardium, followed by imaging the expression of the reporter gene with intravenous [
18
F]-FHBG. The total myocardial [
18
F]-FHBG accumulation was quantified in percent of injected dose (%ID). Immunohistochemistry and autoradiography demonstrated HSV1-sr39tk enzyme (HSV1-sr39TK) and accumulation of [
18
F]-FHBG in the inoculated myocardium in 3 rats each. In 24 rats with various viral titers, the %ID was correlated with ex vivo well counting (
r
2
=0.981,
P
<0.0001) and myocardial HSV1-sr39TK activity by tissue enzyme activity assay (
r
2
=0.790,
P
<0.0001). Myocardial [
18
F]-FHBG accumulation was identified at viral titers down to 1×10
7
pfu. In 6 rats serially imaged up to day 17, myocardial [
18
F]-FHBG accumulation on microPET peaked on days 3 to 5 and was no longer identified on days 10 to 17.

Conclusions—

HSV1-sr39tk reporter gene expression can be monitored with [
18
F]-FHBG and microPET in rat myocardium quantitatively and serially with high detection sensitivity. Cardiac PET reporter gene imaging offers the potential of monitoring the expression of therapeutic genes in cardiac gene therapy.
C-Reactive Protein and Ischemia in Users and Nonusers of β-Blockers and Statins
Authors: Beattie Mary S. , Shlipak Michael G. , Liu Haiying , Browner Warren S. , Schiller Nelson B. , Whooley Mary A.
Published: January 2003

Background—

Elevated levels of C-reactive protein (CRP) are associated with an increased risk of coronary events, but whether inflammation is associated with inducible ischemia in patients with stable coronary disease is unknown.

Methods and Results—

We recruited patients with known coronary disease from 2 VA Medical Centers and 1 University-based medical center for the Heart and Soul Study. We measured CRP levels in 118 participants who had exercise-induced ischemia and in 111 who did not have inducible ischemia, as determined by stress echocardiography. We used logistic regression to examine the risk of exercise-induced ischemia associated with elevated CRP. We found that 75% (39/52) of participants in the highest CRP category (>0.38 mg/dL) had inducible ischemia, compared with 45% (79/177) in the lower 4 categories combined (adjusted odds ratio 4.2; 95% confidence interval 1.6 to 11;
P
=0.004). However, this association differed in users and nonusers of β-blockers and statins. Among 89 participants who did not use β-blockers, 93% in the highest CRP category had exercise-induced ischemia, compared with 42% in the lower 4 categories (
P
=0.03). Among 67 participants who did not use statins, 94% in the highest CRP category had exercise-induced ischemia, compared with 44% in the lower 4 categories (
P
=0.009). We did not observe a significant association between CRP and ischemia among participants who were treated with either of these medications.

Conclusion—

Elevated CRP levels are associated with inducible ischemia in patients with stable coronary disease, particularly among those not treated with β-blockers or statins.
Increased Medial Degradation With Pseudo-Aneurysm Formation in Apolipoprotein E–Knockout Mice Deficient in Tissue Inhibitor of Metalloproteinases-1
Authors: Lemaître Vincent , Soloway Paul D. , D’Armiento Jeanine
Published: January 2003

Background—

The tissue inhibitor of metalloproteinases-1 (TIMP-1) is expressed in atherosclerotic lesions, where it may play a critical role in regulating the activity of matrix metalloproteinases (MMPs). Several MMPs are overexpressed in the atherosclerotic plaque, and they are believed to contribute to the expansion and rupture of the lesion.

Methods and Results—

The
Timp-1
–knockout mouse model (
Timp-1
−/−
) was crossed into the apolipoprotein E–knockout (apoE0) background. A study population of male apoE0 mice, half of them deficient in TIMP-1, was fed an atherogenic diet. After 10 weeks of the diet, the mean lesion sizes of the two groups of animals were not significantly different, and the average content of fibrillar collagen and macrophages in the lesions was similar. There was no sign of plaque hemorrhage, even after 22 weeks of high-fat diet, indicating that deficiency in TIMP-1 does not predispose to luminal rupture. However the atherosclerotic lesions of the
Timp-1
−/0
mice developed more aortic medial ruptures, in which all elastic lamellae of the media were degraded and infiltrated with macrophages, forming pseudo-microaneurysms. After 10 weeks of high-fat diet, the
Timp-1
−/0
/apoE0 mice averaged 1.9±1.2 medial ruptures in the proximal aorta, compared with 0.5±0.7 for the apoE0 controls (
P
<0.003). At the site of degradation, in situ zymography revealed that the gelatinolytic activity, mainly associated with macrophages, could be abolished by the addition of MMP inhibitors.

Conclusions—

These data strongly suggest that TIMP-1 plays a key role in preventing medial degradation associated with atherosclerosis through its ability to inhibit the MMPs that are involved in the disruption of the media.
Multiple Infections and Subsequent Cardiovascular Events in the Heart Outcomes Prevention Evaluation (HOPE) Study
Authors: Smieja Marek , Gnarpe Judy , Lonn Eva , Gnarpe Håkan , Olsson Gunnar , Yi Qilong , Dzavik Vladimir , McQueen Matthew , Yusuf Salim
Published: January 2003

Background—

Limited prospective epidemiological data are available on the relation between exposure to
Chlamydia pneumoniae
,
Helicobacter pylori
, cytomegalovirus (CMV), and hepatitis A virus (HAV), individually or as a total pathogen score, and human cardiovascular (CV) disease.

Methods and Results—

We analyzed enrollment sera from 3168 Canadian patients in the Heart Outcomes Prevention Evaluation (HOPE) study for antibodies to
C pneumoniae
,
H pylori
, CMV, and HAV and measured the relation between serostatus and 494 adjudicated trial outcomes of myocardial infarction, stroke, or CV death over 4.5 years of follow-up. CV events were associated with CMV serostatus (covariate-adjusted hazard ratio [HR], 1.24; 95% CI, 1.01, 1.53). Neither
C pneumoniae
IgG (adjusted HR, 0.87; 95% CI, 0.68, 1.10),
C pneumonia
IgA (adjusted HR, 1.10; 95% CI, 0.90, 1.34),
H pylori
IgG (HR, 0.99; 95% CI, 0.82, 1.19), nor HAV IgG (HR, 1.01; 95% CI, 0.83, 1.24) predicted CV events. Total pathogen score was associated with CV events (adjusted HR for 4 versus 1 or 0=1.41; 95% CI, 1.02, 1.96).

Conclusions—

Exposure to CMV but not to
C pneumoniae
,
H pylori
, or HAV was associated with a slight excess risk of subsequent myocardial infarction, stroke, or CV death in HOPE study patients, and total pathogen score based on these infections predicted a small increased hazard of CV events.
Effect of Adrenergic Stimulation on Action Potential Duration Restitution in Humans
Authors: Taggart Peter , Sutton Peter , Chalabi Zaid , Boyett Mark R. , Simon Ron , Elliott Donna , Gill Jaswinder S.
Published: January 2003

Background—

Enhanced sympathetic activity facilitates complex ventricular arrhythmias and fibrillation. The restitution properties of action potential duration (APD) are important determinants of electrical stability in the myocardium. Steepening of the slope of APD restitution has been shown to promote wave break and ventricular fibrillation. The effect of adrenergic stimulation on APD restitution in humans is unknown.

Methods and Results—

Monophasic action potentials were recorded from the right ventricular septum in 18 patients. Standard APD restitution curves were constructed at 3 basic drive cycle lengths (CLs) of 600, 500, and 400 ms under resting conditions and during infusion of isoprenaline (15 patients) or adrenaline (3 patients). The maximum slope of the restitution curves was measured by piecewise linear regression segments of sequential 40-ms ranges of diastolic intervals in steps of 10 ms. Under control conditions, the maximum slope was steeper at longer basic CLs; eg, mean values for the maximum slope were 1.053±0.092 at CL 600 ms and 0.711±0.049 at CL 400 ms (±SEM). Isoprenaline increased the steepness of the maximum slope of APD restitution, eg, from a maximum slope of 0.923±0.058 to a maximum slope of 1.202±0.121 at CL 500 ms. The effect of isoprenaline was greater at the shorter basic CLs. A similar overall effect was observed with adrenaline.

Conclusions—

The adrenergic agonists isoprenaline and adrenaline increased the steepness of the slope of the APD restitution curve in humans over a wide range of diastolic intervals. These results may relate to the known effects of adrenergic stimulation in facilitating ventricular fibrillation.
Altered Temporal Profile of Heat Shock Factor 1 Phosphorylation and Heat Shock Protein 70 Expression Induced by Heat Shock in Nucleus Tractus Solitarii of Spontaneously Hypertensive Rats
Authors: Chan Samuel H.H. , Wang Ling-Lin , Chang Kuei-Feng , Ou Chen-Chun , Chan Julie Y.H.
Published: January 2003

Background—

We demonstrated recently that heat shock (HS)–induced heat shock protein 70 (HSP70) expression in bilateral nucleus tractus solitarii (NTS), the terminal site in the brain stem for primary baroreceptor afferents, confers cardiovascular protection against heatstroke by potentiating baroreceptor reflex (BRR) response. This study evaluated the hypothesis that altered regulation of HSP70 expression may be associated with the heightened susceptibility to heatstroke during hypertension.

Methods and Results—

Spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats anesthetized with propofol were used. Compared with WKY rats, significant induction in HSP70 or phosphorylation of heat shock factor 1 (HSF1), but not HSF2, in the NTS and potentiation of BRR response in SHR occurred earlier (4 versus 8 hours), reaching peak magnitude sooner (16 versus 24 hours), and declined more rapidly after a brief hyperthermic HS (42±0.5°C for 15 minutes). The protection conferred by HS against hypotension and bradycardia during the onset of heatstroke (45°C for 60 minutes), although effective, was less effective in SHR. Microinjection bilaterally into the NTS of the selective protein kinase A (PKA) inhibitor H-89 (100 pmol) or the selective PKC inhibitor calphostin C (100 pmol) significantly attenuated all of the above events induced in SHR by HS. However, only H-89 was effective in WKY rats.

Conclusions—

An altered temporal profile of HS-induced HSP70 expression or potentiation of BRR response by concurrent activation via both PKA and PKC pathways of phosphorylation of HSF1 in the NTS may be associated with greater susceptibility to heatstroke during hypertension.
Divergent Nitric Oxide Bioavailability in Men and Women With Sickle Cell Disease
Authors: Gladwin Mark T. , Schechter Alan N. , Ognibene Frederick P. , Coles Wynona A. , Reiter Christopher D. , Schenke William H. , Csako Gyorgy , Waclawiw Myron A. , Panza Julio A. , Cannon Richard O.
Published: January 2003

Background—

Although reduced endothelial nitric oxide (NO) bioavailability has been demonstrated in arteriosclerotic vascular disease, the integrity of this system in sickle cell disease remains uncertain.

Methods and Results—

We measured forearm blood flow in 21 patients with sickle cell disease (hemoglobin SS genotype) and 18 black control subjects before and after intra-arterial infusions of acetylcholine, nitroprusside, and the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA). Endothelium-dependent vasodilation, measured by the percent increase in flow induced by acetylcholine infusion, was significantly greater than in controls (252±37% for patients versus 134±24% for controls;
P
<0.0001). However, there was a large sex difference in blood flow responses between female and male patients (340±46% versus 173±41%;
P
=0.035). Similarly, basal NO bioactivity, as measured by the percent decrease in flow induced by L-NMMA, was depressed in male compared with female patients (−17±5% versus −34±4%;
P
=0.01), as was the response to nitroprusside (86±21% versus 171±22%;
P
=0.008). L-NMMA reduced the blood flow response to acetylcholine in women, but not in men. Sex differences in vascular cell adhesion molecule-1 were appreciated, with significant correlations between levels of soluble vascular cell adhesion molecule-1 and blood flow responses to L-NMMA and nitroprusside (
r
=0.53,
P
=0.004 and
r
=−0.66,
P
<0.001, respectively).

Conclusions—

NO bioavailability and NO responsiveness are greater in women than in men with sickle cell disease and determines adhesion molecule expression. Endothelium-dependent blood flows are largely non-NO mediated in male patients. These results provide a possible mechanism for reported sex differences in sickle cell disease morbidity and mortality and provide a basis for novel pharmacological interventions.
Randomized Evaluation of the Safety and Efficacy of Enoxaparin Versus Unfractionated Heparin in High-Risk Patients With Non-ST-Segment Elevation Acute Coronary Syndromes Receiving the Glycoprotein IIb/IIIa Inhibitor Eptifibatide
Authors: Goodman Shaun G. , Fitchett David , Armstrong Paul W. , Tan Mary , Langer Anatoly
Published: January 2003

Background—

Current pharmacotherapeutic options for high-risk non-ST-segment elevation acute coronary syndrome patients include aspirin, clopidogrel, heparin, and platelet glycoprotein IIb/IIIa inhibition. A key issue of uncertainty is the safety and efficacy of combination glycoprotein IIb/IIIa inhibitor and low-molecular-weight heparin therapy.

Methods and Results—

We randomized 746 patients with rest ischemic discomfort within 24 hours after the onset of symptoms and ST-segment deviation and/or elevation of serum cardiac markers to receive open-label enoxaparin (1 mg/kg subcutaneously twice daily) or unfractionated heparin (70-U/kg bolus; 15 U · kg
−1
· h
−1
infusion, titrated to an activated partial thromboplastin time of 1.5 to 2 times control) for 48 hours. All patients received aspirin and eptifibatide (180-μg/kg bolus; 2 μg · kg
−1
· min
−1
infusion). Major non-coronary artery bypass surgery-related bleeding at 96 hours (primary safety outcome) was significantly lower among enoxaparin-treated patients than among heparin-treated patients (1.8% versus 4.6%,
P
=0.03). Minor bleeding was more frequent in the enoxaparin group (30.3% versus 20.8%,
P
=0.003). Patients in the enoxaparin group were less likely to experience ischemia as detected by continuous ECG evaluation (primary efficacy outcome) during the initial (14.3% versus 25.4%,
P
=0.0002) and subsequent (12.7% versus 25.9%,
P
<0.0001) 48-hour monitoring periods. Death or myocardial infarction at 30 days was significantly lower in the enoxaparin group (5% versus 9%,
P
=0.031).

Conclusions—

When aspirin and eptifibatide are used in high-risk non-ST-segment elevation acute coronary syndrome patients, enoxaparin improves outcomes (determined on the basis of better safety and efficacy) compared with currently recommended unfractionated heparin therapy and provides a useful novel alternative therapeutic strategy.
Age-Dependent Impairment of Reendothelialization After Arterial Injury
Authors: Gennaro Giuseppa , Ménard Catherine , Michaud Sophie-Élise , Rivard Alain
Published: January 2003

Background—

The mechanisms responsible for the association between advanced age and atherosclerotic diseases are not clear. Because atherosclerosis develops in response to local endothelial injuries, we investigated the effect of aging on vascular healing and reendothelialization.

Methods and Results—

Endothelium denudation was performed by balloon angioplasty of the iliac arteries in young and old New Zealand White rabbits. Planimetric analysis after Evans Blue staining at 28 days after injury showed a significant decrease in reendothelialization in old versus young animals, which was associated with an important increase in neointimal formation in old rabbits. Vascular endothelial growth factor (VEGF) was rapidly induced after balloon injury. However, arterial VEGF expression was significantly reduced in old versus young animals. To confirm the role of VEGF in the age-dependent impairment of reendothelialization, an adenoviral vector encoding for VEGF
165
(adeno-VEGF) was locally delivered at the time of iliac artery angioplasty. Compared with animals treated with the control vector (adeno-βGal), reendothelialization was significantly improved and neointimal formation reduced in old rabbits treated with adeno-VEGF.

Conclusions—

These results document for the first time an age-dependent impairment of reendothelialization after arterial injury. Our study indicates that VEGF supplementation may represent a useful strategy to accelerate reendothelialization and improve vascular healing in the context of aging.
Beneficial Effects of Phosphodiesterase 5 Inhibition in Pulmonary Hypertension Are Influenced by Natriuretic Peptide Activity
Authors: Zhao Lan , Mason Nicola A. , Strange Julian W. , Walker Hamish , Wilkins Martin R.
Published: January 2003

Background—

Phosphodiesterase type 5 (PDE5) inhibitors (eg, sildenafil) are a novel, orally active approach to the treatment of pulmonary arterial hypertension. The role of natriuretic peptides in the response to sildenafil was examined in mice lacking NPR-A, a guanylyl cyclase-linked natriuretic peptide receptor, in which pulmonary hypertension was induced by hypoxia.

Methods and Results—

Mice homozygous for NPR-A (
NPR-A
+/+
) and null mutants (
NPR-A
−/−
) were studied. Sildenafil inhibited the pressor response to acute hypoxia in the isolated perfused lungs of both genotypes. This effect was greater in the presence of atrial natriuretic peptide in the perfusate in
NPR-A
+/+
mice but not
NPR-A
−/−
animals. In vivo, NPR-A mutants had higher basal right ventricular (RV) systolic pressures (RVSPs) than did
NPR-A
+/+
mice, and this was not affected by 3 weeks of treatment with sildenafil (25 mg · kg
−1
· d
−1
). Both genotypes exhibited a rise in RVSP and RV weight with chronic hypoxia (10% O
2
for 21 days); RVSP and RV weight were reduced by continuous sildenafil administration in
NPR-A
+/+
mice, but only RVSP showed evidence of a response to the drug in
NPR-A
−/−
mice. The effect of sildenafil on hypoxia-induced pulmonary vascular muscularization and cyclic GMP levels was also blunted in
NPR-A
−/−
mice.

Conclusions—

The natriuretic peptide pathway influences the response to PDE5 inhibition in hypoxia-induced pulmonary hypertension, particularly its effects on RV hypertrophy and vascular remodeling.
Mortality among Farmers and Cotters in the Sundsvall Area of Sweden During the Nineteenth Century
Authors: Curtis Stephan Michael
Published: January 2003

During the latter half of the nineteenth century, the Sundsvall area of Sweden underwent a dramatic economic transformation powered by the exceptional growth of the sawmill industry. Industrialization certainly had devastating consequences for the health of sawmill employees and their families but also contributed to a gradual reduction in mortality rates among many farmers and cotters. This was particularly true among those who could benefit from the increased market for their produce found in the town and near the sawmills. Much of this study examines the consequences of industrial growth on the population's health. Nonetheless, the marked increase in tuberculosis mortality among women toward the end of the century illuminates the need to consider the social and cultural implications of industrialization rather than simply its economic characteristics when examining mortality patterns.
Family Ideology and the Charity Organization Society in Great Britain During the First World War
Authors: Gente Magali
Published: January 2003

The Charity Organization Society, which became the Family Welfare Association in 1946, was turned from a philanthropic institution born in the nineteenth century into a useful social structure under the double influence of the Great War and the Radical Liberals' family ideology. The huge development of state intervention and the government's quest for general improvement in family life, in the name of imperialistic defense, national efficiency, and social harmony, entailed a symbiotic cooperation between private help and public care and illustrated the construction of a family ideal in Great Britain both by social policy makers and the voluntary movement.
Family War Experience and Later-Life Political Activism among Okinawan Women
Authors: Schultz Kristen
Published: January 2003

Using interviews conducted by the author in 1997 and 1998 with forty-seven Okinawan women born between 1919 and 1980, this article examines, from a lifecourse perspective, the effects of childhood or family World War II experience on activism in the anti–U.S. military peace movement. How does the memory of war and the historical context of early adulthood shape later-life activism in peace movements? The research examines links between the historical context of early adulthood, personal and family war experiences, and later-life political activism. Women reaching early adulthood in the immediate postwar period in Okinawa are argued to be more active in the antimilitary political movement than women reaching early adulthood more than thirty years after the end of World War II. Historical context, linked lives, and timing of war experience are considered to be key determinants of political activism.
Philosophical Writings on the Family in Sixteenth- and Seventeenth-Century Europe
Authors: Freedman Joseph S.
Published: January 2003

Recent research on the early modern European family has largely been based on archival sources that are extant for relatively few localities during this same period. This research can be augmented by examining discussions of the family contained within academic writings on theology, jurisprudence, medicine, and philosophy during the early modern period. This article focuses on philosophical writings that arose in connection with sixteenth- and seventeenth-century academic instruction. These writings routinely discuss the proper relationship between husband and wife, between parents and children, and between masters and servants; also discussed are various categories of domestic possessions and how these possessions should be acquired and administered. Within these philosophical writings, one controversial issue pertaining to family life is sometimes raised: whether servants are more essential to the family than children. These writings uniformly equate the family with the nuclear family; in doing so, they provide collaboration for similar findings by social and demographic historians.
The Eye of the Follower
Authors: Nye Judith L.
Published: January 2003

The effects of inference-based and recognition-based processes on attributions of leadership responsibility were examined. Participants (126 women, 42 men) competed in small groups in this 2 (performance: win vs. lose) 2 (material sabotage: sabotaged vs. not sabotaged) design study. A third factor addressed how well group leaders matched participants’leader-ship prototypes. Winning leaders were attributed more responsibility for the group outcome than losing leaders; also, sabotage served as a discounting cue, and losing leaders of sabotaged groups were held less responsible. Unexpectedly, no prototype matching was revealed. Participants’ attributions regarding responsibility of the group and self were similar to leader attributions, suggesting that followers’ perceptions differ from those of outside observers. Thus, leaders may not be the only cause of group outcomes in the minds of actual followers.
12-Lipoxygenase Pathway Increases Aldosterone Production, 3′,5′-Cyclic Adenosine Monophosphate Response Element-Binding Protein Phosphorylation, and p38 Mitogen-Activated Protein Kinase Activation in H295R Human Adrenocortical Cells
Authors: Gu Jiali , Wen Yeshao , Mison Angeles , Nadler Jerry L.
Published: January 2003

Evidence suggests that the 12-lipoxygenase (LO) pathway mediates angiotensin II (Ang II)-induced aldosterone synthesis in adrenal glomerulosa cells. To study the mechanisms of 12-LO pathway on aldosterone synthesis, the human adrenocortical cell line, H295R, was transiently transfected with a mouse leukocyte type of 12-LO. Overexpression of 12-LO stimulated aldosterone production 2.7-fold as well as the reporter gene activity of CYP11B2 gene-encoding human aldosterone synthase by 5-fold over that in mock-transfected cells. Ang II further enhanced aldosterone production, which could be blocked by a 12-LO inhibitor, baicalein, in mock cells and cells overexpressing 12-LO. Ang II stimulated cAMP response element-binding protein (CREB) phosphorylation in a dose- and time-dependent fashion in parent H295R cells. Overexpression of 12-LO increased phosphorylation of CREB/activating transcription factor (ATF)-1 1.5-fold over that in mock cells under basal conditions. Ang II led to a further 5.2- and 7.5-fold increase in mock cells and 12-LO cells, respectively. Overexpression of 12-LO induced p38 MAPK activation. The 12-LO product, 12-hydroxyeicosatetraenoic acid, increased phosphorylation of CREB/ATF-1 3.6-fold and phosphorylation of p38 MAPK 8-fold over basal. The p38 MAPK inhibitor SB203580 inhibited Ang II- and 12-LO pathway-induced phosphorylated CREB/ATF-1, suggesting a role of p38 MAPK in Ang II and 12-LO pathway signaling. These results suggest that 12-LO stimulation leads to aldosterone production in H295R cells in part through activation of CREB/ATF-1 and p38 MAPK pathway.
Proliferative Phase Sertoli Cells Display a Developmentally Regulated Response to Activin in Vitro
Authors: Buzzard Jeremy J. , Farnworth Paul G. , de Kretser David M. , O’Connor Anne E. , Wreford Nigel G. , Morrison John R.
Published: January 2003

We have used cultures of highly purified, proliferating rat Sertoli cells collected from d 3, 6, and 9 rat pups to investigate the role of activin A on Sertoli cell division. These studies demonstrate that activin A acts directly on d 6 and 9, but not d 3, Sertoli cells to induce proliferation, both alone and synergistically with FSH. In addition to stimulating proliferation, activin A induces secretion of inhibins A and B as determined by specific ELISAs. We demonstrate that the synergy between activin A and FSH is not due to local actions of secreted inhibin or follistatin. We have used real-time fluorometric RT-PCR to demonstrate that activin regulates expression of activin receptor and follistatin mRNA by Sertoli cells. Saturation binding studies using 125I-activin A indicate that synergy between activin and FSH may be due to increased numbers of activin receptors on the Sertoli cell. Finally, we show that activin A was secreted at high levels by cultured peritubular cells but was undetectable in high purity proliferating Sertoli cell cultures, suggesting that activin A functions as a paracrine factor during postnatal testis development.
Dronerarone Acts as a Selective Inhibitor of 3,5,3′-Triiodothyronine Binding to Thyroid Hormone Receptor-α1: In Vitro and in Vivo Evidence
Authors: van Beeren H. C. , Jong W. M. C. , Kaptein E. , Visser T. J. , Bakker O. , Wiersinga W. M.
Published: January 2003

Dronedarone (Dron), without iodine, was developed as an alternative to the iodine-containing antiarrhythmic drug amiodarone (AM). AM acts, via its major metabolite desethylamiodarone, in vitro and in vivo as a thyroid hormone receptor α1 (TRα1) and TRβ1 antagonist. Here we investigate whether Dron and/or its metabolite debutyldronedarone inhibit T3 binding to TRα1 and TRβ1in vitro and whether dronedarone behaves similarly to amiodarone in vivo.

In vitro , Dron had a inhibitory effect of 14% on the binding of T3 to TRα1, but not on TRβ1. Desethylamiodarone inhibited T3 binding to TRα1 and TRβ1 equally. Debutyldronedarone inhibited T3 binding to TRα1 by 77%, but to TRβ1 by only 25%.

In vivo , AM increased plasma TSH and rT3, and decreased T3. Dron decreased T4 and T3, rT3 did not change, and TSH fell slightly. Plasma total cholesterol was increased by AM, but remained unchanged in Dron-treated animals. TRβ1-dependent liver low density lipoprotein receptor protein and type 1 deiodinase activities decreased in AM-treated, but not in Dron-treated, animals. TRα1-mediated lengthening of the QTc interval was present in both AM- and Dron-treated animals.

The in vitro and in vivo findings suggest that dronedarone via its metabolite debutyldronedarone acts as a TRα1-selective inhibitor.
Selection of the Dominant Follicle and Insulin-Like Growth Factor (IGF)-Binding Proteins: Evidence that Pregnancy-Associated Plasma Protein A Contributes to Proteolysis of IGF-Binding Protein 5 in Bovine Follicular Fluid
Authors: Rivera G. M. , Fortune J. E.
Published: January 2003

Development of a dominant follicle is associated with decreased intrafollicular low molecular weight IGF-binding proteins (namely IGFBP-2, -4, and -5) and increased proteolysis of IGFBP-4 by pregnancy-associated plasma protein A (PAPP-A). In addition to IGFBP-4 proteolytic activity, bovine follicular fluid contains strong proteolytic activity for IGFBP-5, but not for IGFBP-2. Here we show that the IGFBP-5 protease present in bovine follicular fluid is a neutral/basic pH-favoring, Zn2+ metalloprotease very similar to the previously described IGFBP-4 protease. We hypothesized that immunoneutralization and immunoprecipitation with anti-PAPP-A antibodies would result in abrogation of the IGFBP-4, but not the IGFBP-5, proteolytic activity in follicular fluid. As expected, anti-PAPP-A antibodies were able to neutralize and precipitate the IGFBP-4, but not the IGFBP-5, proteolytic activity of human pregnancy serum, which was used as a positive control for PAPP-A. Surprisingly, immunoneutralization and immunoprecipitation of follicular fluid from bovine preovulatory follicles with anti-PAPP-A antibodies abrogated both IGFBP-4 and IGFBP-5 proteolysis. Quantitative results derived from phosphorimaging revealed a complete inhibition of both IGFBP-4 and -5 proteolysis by follicular fluid incubated for 2 or 5 h in the presence of anti-PAPP-A antibodies. After 18 h of incubation, anti-PAPP-A antibodies still inhibited IGFBP-5 degradation, although with an efficiency lower than that for IGFBP-4 degradation. Both proteolytic activities have identical electrophoretic mobility, and a single band (∼400 kDa) was detected by Western immunoblotting of bovine follicular fluid with anti-PAPP-A antibodies. Proteolysis of IGFBP-5 was readily detectable in follicular fluid from dominant follicles and was negligible in subordinate follicles from the same cohort. These results suggest that an active intrafollicular IGFBP-4/-5 proteolytic system, in which PAPP-A is the major protease involved, is an important determinant of follicular fate.
Statins Augment Vascular Endothelial Growth Factor Expression in Osteoblastic Cells via Inhibition of Protein Prenylation
Authors: Maeda Toyonobu , Kawane Tetsuya , Horiuchi Noboru
Published: January 2003

Statins such as simvastatin are 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors that inhibit cholesterol synthesis. We presently investigated statin effects on vascular endothelial growth factor (VEGF) expression in osteoblastic cells. Hydrophobic statins including simvastatin, atorvastatin, and cerivastatin–but not a hydrophilic statin, pravastatin–markedly increased VEGF mRNA abundance in nontransformed osteoblastic cells (MC3T3-E1). Simvastatin (10−6m) time-dependently augmented VEGF mRNA expression in MC3T3-E1 cells, mouse stromal cells (ST2), and rat osteosarcoma cells (UMR-106). According to heterogeneous nuclear RNA and Northern analyses, 10−6m simvastatin stimulated gene expression for VEGF in MC3T3-E1 cells without altering mRNA stability. Transcriptional activation of a VEGF promoter-luciferase construct (−1128 to +827), significantly increased by simvastatin administration. As demonstrated by gel mobility shift assay, simvastatin markedly enhanced the binding of hypoxia-responsive element-protein complexes. These results indicate that the stimulation of the VEGF gene by simvastatin in MC3T3-E1 cells is transcriptional in nature. VEGF secretion into medium was increased in MC3T3-E1 by 10−6m simvastatin. Pretreating MC3T3-E1 cells with mevalonate or geranylgeranyl pyrophosphate, a mevalonate metabolite, abolished simvastatin-induced VEGF mRNA expression; manumycin A, a protein prenylation inhibitor, mimicked statin effects on VEGF expression. The effect of simvastatin was blocked by pretreatment with wortmannin and LY294002, specific phosphatidylinositide-3 kinase inhibitors. Simvastatin enhanced mineralized nodule formation in culture, whereas coincubation with mevalonate, geranylgeranyl pyrophosphate, LY294002, or VEGF receptor 2 inhibitor (SU1498) abrogated statin-induced mineralization. Thus, statins stimulate VEGF expression in osteoblasts via reduced protein prenylation and the phosphatidylinositide-3 kinase pathway, promoting osteoblastic differentiation.
Extracellular Signal-Regulated Kinase and c-Src, But Not Jun N-Terminal Kinase, Are Involved in Basal and Gonadotropin-Releasing Hormone-Stimulated Activity of the Glycoprotein Hormone α-Subunit Promoter
Authors: Harris Dagan , Chuderland Dana , Bonfil David , Kraus Sarah , Seger Rony , Naor Zvi
Published: January 2003

Addition of a GnRH agonist (GnRH-A) to αT3-1 cells stimulates different MAPK cascades: ERK, Jun N-terminal kinase (JNK), and p38. Activation of JNK, ERK, and p38 shows a unique fold activation ratio of 25:12:2, which might encode signal specificity. ERK is translocated to the nucleus within 20 min with a peak at 120 min of GnRH-A stimulation. We used the human α-subunit promoter linked to chloramphenicol acetyl transferase (αCAT) to examine the role of ERK, JNK, and c-Src, which is implicated in MAPK activation, in basal and GnRH-stimulated αCAT. Addition of GnRH-A resulted in a 3-fold increase in αCAT, whereas the Ca2+ ionophore ionomycin and the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA) had no effect. Addition of GnRH-A and TPA, but not GnRH-A and ionomycin, produced a synergistic response, whereas removal of Ca2+, but not down-regulation of TPA-sensitive PKCs, abolished GnRH-A-stimulated αCAT. Thus, regulation of α-promoter activity by GnRH is Ca2+ dependent and is further augmented by PKC. Cotransfection of αCAT and constitutively active or dominant negative plasmids of ERK and JNK cascade members, or the use of the ERK inhibitor PD98059, revealed that ERK, but not JNK, is involved in basal and GnRH-A-stimulated αCAT. Because c-Src participates in MAPK activation by GnRH, we also studied its role. Cotransfection of αCAT and the dominant negative form of c-Src or incubation with the c-Src inhibitor PP1 reduced GnRH-A-stimulated αCAT. The 5′-deletion analysis revealed that the −846/−420 region participated in basal α-transcription. In addition, the −346/−156 region containing the pituitary glycoprotein hormone basal element, α-basal elements, glycoprotein-specific element, and upstream response element is involved in basal and GnRH-A-stimulated αCAT. ERK contribution to GnRH maps to −346/−280 containing the pituitary glycoprotein hormone basal element and α-basal elements 1/2. Surprisingly, although c-Src is involved in GnRH-A-stimulated ERK, its involvement is mapped to another region (−280/−180) containing the glycoprotein-specific element. Thus, ERK and c-Src but not JNK are involved in basal and GnRH-A-stimulated-αCAT, whereas c-Src contribution is independent of ERK activation.
Rat Anterior Pituitary Folliculostellate Cells Are Targets of Interleukin-1β and a Major Source of Intrapituitary Follistatin
Authors: Bilezikjian Louise M. , Leal Angela M. O. , Blount Amy L. , Corrigan Anne Z. , Turnbull Andrew V. , Vale Wylie W.
Published: January 2003

Folliculostellate cells of the anterior pituitary are postulated to be an important source of factors, such as follistatin, that regulate pituitary function by intercellular communication. To gain further insight into the function of this cell type, folliculostellate cells were enriched from cultured rat anterior pituitary cells, and an immortalized cell line designated FS/D1h was established and characterized. These FS/D1h cells express S100 immunoreactivity and produce IL-6 but not pituitary hormones such as GH, ACTH, FSH, and LH. Importantly, FS/D1h cells express large amounts of follistatin mRNA and secrete the protein, as quantified indirectly by the amount of [125I]activin A immunoprecipitated with a follistatin antiserum. The FS/D1h cells also express α, βA, and βB inhibin/activin subunit mRNAs, but whether they produce the corresponding activins and inhibins has not been determined. The response of FS/D1h cells to agents thought to modulate folliculostellate cell function was evaluated. IL-1β (0.005–5 nm) stimulated the secretion of follistatin and increased mRNA expression. In parallel, IL-6 secretion was stimulated. Dexamethasone, pituitary adenylate cyclase-activating polypeptide(1–27), and lipopolysaccharide but not testosterone, 12-O-tetradecanoylphorbol-13-acetate, or forskolin also increased follistatin secretion. Surprisingly, activin had no effect on follistatin mRNA levels, despite the fact that FS/D1h cells express ActRII, ActRIIB, and ALK-4 (ActRIB). Activin, on the other hand, induced Smad7 mRNA accumulation and exerted an antiproliferative effect on FS/D1h cells. Altogether, these observations support the possibility that follistatin originating from folliculostellate cells participates in mediating the effects of IL-1β, glucocorticoids, and other agents on the response of pituitary cells to activins.
5α-Androstane-3α,17β-Diol Is Formed in Tammar Wallaby Pouch Young Testes by a Pathway Involving 5α-Pregnane-3α,17α-Diol-20-One as a Key Intermediate
Authors: Wilson Jean D. , Auchus Richard J. , Leihy Michael W. , Guryev Oleg L. , Estabrook Ronald W. , Osborn Susan M. , Shaw Geoffrey , Renfree Marilyn B.
Published: January 2003

The synthetic pathway by which 5α-androstane-3α,17β-diol (5α-adiol) is formed in the testes of tammar wallaby pouch young was investigated by incubating testes from d 20–40 males with various radioactive precursors and analyzing the metabolites by thin-layer chromatography and HPLC. [3H]Progesterone was converted to 17-hydroxyprogesterone, which was converted to 5α-adiol by two pathways: One involves the formation of testosterone and dihydrotestosterone as intermediates, and the other involves formation of 5α-pregnane-3α,17α-diol-20-one (5α-pdiol) and androsterone as intermediates. Formation of 5α-adiol from both [3H]testosterone and [3H]progesterone was blocked by the 5α-reductase inhibitor 4MA. The addition of nonradioactive 5α-pdiol blocked the conversion of [3H]progesterone to 5α-adiol, and [3H]5α-pdiol was efficiently converted to androsterone and 5α-adiol. We conclude that expression of steroid 5α-reductase in the developing wallaby testes allows formation of 5α-reduced androgens by a pathway that does not involve testosterone as an intermediate.
Global Transcription Profiling of Estrogen Activity: Estrogen Receptor α Regulates Gene Expression in the Kidney
Authors: Jelinsky Scott A. , Harris Heather A. , Brown Eugene L. , Flanagan Kathryn , Zhang Xiaochun , Tunkey Christopher , Lai KehDih , Lane Malcolm V. , Simcoe Donna K. , Evans Mark J.
Published: January 2003

Estrogen receptors (ERs) are expressed in numerous organs, although only a few organs are considered classical targets for estrogens. We have completed a systematic survey of estrogen regulation of approximately 10,000 genes in 13 tissues from wild-type and ERβΚΟ mice treated sc with vehicle or 17β-estradiol (E2) for 6 wk. The uterus and pituitary had the greatest number of genes regulated by E2, whereas the kidney had the third largest number of regulated genes. In situ hybridizations localized E2 regulation in the kidney to the juxtamedullary region of the cortex in both the mouse and rat. The ED50 for gene inductions in the kidney was 3 μg/kg·d, comparable with the 2.4 μg/kg·d ED50 for c-fos induction in the uterus. E2 regulations in the kidney were intact in ERβKO mice, and the ERα-selective agonist propylpyrazole triol acted similarly to E2, together suggesting an ERα-mediated mechanism. Several genes were induced within 2 h of E2 treatment, suggesting a direct activity of ERα within the kidney. Finally, the combination of the activation function (AF)1-selective agonist tamoxifen plus ERαKOCH mice expressing an AF1-deleted version of ERα allowed delineation of genes with differing requirements for AF1 or AF2 activity in the kidney.
Involvement of Cyclic Adenosine 3′,5′-Monophosphate in the Differential Regulation of Activin βA and βB Expression by Gonadotropin in the Zebrafish Ovarian Follicle Cells
Authors: Wang Yajun , Ge Wei
Published: January 2003

Activin is a dimeric protein consisting of two similar but distinct β-subunits, βA and βB. In our previous studies, both activin A (βAβA) and activin B (βBβB) have been demonstrated to stimulate oocyte maturation and promote oocyte maturational competence in the zebrafish. Follistatin, a specific activin-binding protein, can block both activin- and gonadotropin-induced final oocyte maturation in vitro, suggesting that activin is likely a downstream mediator of gonadotropin actions in the zebrafish ovary. In the present study, a full-length cDNA encoding zebrafish ovarian activin βA was cloned and sequenced. The precursor of zebrafish activin βA consists of 395 amino acids and its mature region exhibits about 78% homology with that of mammals. Using an in vitro primary culture of the ovarian follicle cells and semiquantitative RT-PCR assays, we examined the regulation of activin βA and βB expression by human chorionic gonadotropin (hCG) and its intracellular signal transduction mechanisms. hCG (15 IU/ml) increased the mRNA level of activin βA-subunit; however, it significantly down-regulated the steady-state expression level of activin βB in a time- and dose-dependent manner. The differential regulation of the two β-subunits by hCG could be mimicked by 3-isobutyl-1-methylxanthine, forskolin, and dibutyryl-cAMP, suggesting involvement of the intracellular cAMP pathway. Interestingly, H89 (a specific inhibitor of protein kinase A, PKA) could effectively block hCG- and forskolin-stimulated activin βA expression at 10 μm, but it was unable to reverse the inhibitory effects of hCG and forskolin on βB expression. This suggests that the hCG-stimulated activin βA expression is dependent on the activation of the cAMP-PKA pathway, whereas the inhibitory effect of hCG on activin βB expression is likely mediated by PKA-independent pathway(s).
Biological Activity of 17β-Estradiol-3-Sulfate in Ovine Fetal Plasma and Uptake in Fetal Brain
Authors: Wood Charles E. , Gridley Kelly E. , Keller-Wood Maureen
Published: January 2003

In sheep, the fetal hypothalamus-pituitary-adrenal axis plays a central role in the initiation of parturition. We have reported that estradiol dramatically increases the activity of the fetal hypothalamus-pituitary-adrenal (HPA) axis. Sulfoconjugated estrogens are known to circulate in high concentrations in fetal plasma. We have reported the expression and abundant activity of steroid sulfatase within the fetal brain regions important for HPA axis control, and we have proposed that sulfoconjugated estrogens in fetal plasma are deconjugated (and therefore converted to a biologically active form) in fetal brain. The present study was designed to test the hypothesis that exogenous estradiol-3-sulfate stimulates HPA axis activity in late gestation fetal sheep and that it is concentrated by fetal brain tissue. We infused estradiol-3-sulfate iv into fetal sheep (125–135 d gestation; term = 147 d) at rates of 0, 0.25, and 1.0 mg/d for 5 d and performed serial sampling of fetal blood before and at the end of the infusion periods. Infusions increased fetal plasma estradiol-3-sulfate concentrations and produced dose-related increases in HPA axis activity. The action of the steroid on the fetal brain was also demonstrated as dose-related increases in the abundance of Fos in fetal cerebellum. In a second study we measured the uptake of sulfoconjugated and unconjugated estrogen (estrone-3sulfate and estrone, respectively) into the fetal brain (124–128 d gestation) in vivo. Both forms of estrogen were concentrated in fetal brain, with the uptake of estrone greater than that of estrone-3-sulfate. We conclude that sulfoconjugated estrogens augment fetal HPA axis activity and that they can cross the fetal blood-brain barrier. We propose that in late gestation the large circulating pool of sulfoconjugated estrogen is a biologically important source of active hormone that might play a role in the timing of parturition in sheep.
Distribution of Vesicular Glutamate Transporter-2 Messenger Ribonucleic Acid and Protein in the Septum-Hypothalamus of the Rat
Authors: Lin Winston , McKinney Kyle , Liu Liansheng , Lakhlani Shruti , Jennes Lothar
Published: January 2003

The excitatory neurotransmitter glutamate is involved in the control of most, perhaps all, neuroendocrine systems, yet the sites of glutamatergic neurons and their processes are unknown. Here, we used in situ hybridization and immunohistochemistry for the neuron-specific vesicular glutamate transporter-2 (VGLUT2) to identify the neurons in female rats that synthesize the neurotransmitter glutamate as well as their projections throughout the septum-hypothalamus. The results show that glutamatergic neurons are present in the septum-diagonal band complex and throughout the hypothalamus. The preoptic area and ventromedial and dorsomedial nuclei are particularly rich in glutamatergic neurons, followed by the supraoptic, paraventricular, and arcuate nuclei, whereas the suprachiasmatic nucleus does not express detectable amounts of VGLUT2 mRNA. Immunoreactive neurites are seen in very high densities in all regions analyzed, particularly in the preoptic region, followed by the ventromedial, dorsomedial, and arcuate nuclei as well as the external layer of the median eminence, whereas the mammillary complex does not exhibit VGLUT2 immunoreactivity. Many VGLUT2 immunoreactive fibers also contained synaptophysin, suggesting that the transporter is indeed localized to presynaptic terminals. Together, the results identify glutamatergic cell bodies throughout the septum-hypothalamus in region-specific patterns and show that glutamatergic nerve terminals are present in very large numbers such that most neurons in these brain regions can receive glutamatergic input. We examined the GnRH system as an example of a typical neuroendocrine system and could show that the GnRH perikarya are closely apposed by many VGLUT2-immunoreactive boutons, some of which also contained synaptophysin. The presence of VGLUT2 mRNA-containing cells in specific nuclei of the hypothalamus indicates that many neuroendocrine neurons coexpress glutamate as neurotransmitter, in addition to neuropeptides. These systems include the oxytocin, vasopressin, or CRH neurons as well as many others in the periventricular and mediobasal hypothalamus. The presence of VGLUT2 mRNA in steroid-sensitive regions of the hypothalamus, such as the anteroventral periventricular, paraventricular, or ventromedial nuclei indicates that gonadal and adrenal steroid can directly alter the functions of these glutamatergic neurons.