bluuebunny/crossref_metadata_2025 huggingface dataset:
Maternal-Fetal Flow, Negative Events, and Preeclampsia
Authors: Mello Giorgio , Parretti Elena , Gensini Francesca , Sticchi Elena , Mecacci Federico , Scarselli Gianfranco , Genuardi Maurizio , Abbate Rosanna , Fatini Cinzia
Published: April 2003

The risk for an adverse pregnancy outcome is markedly higher in women with history of preeclampsia. This may stem from impaired placentation in early gestation and from high impedance to flow in uteroplacental circulation. The renin-angiotensin system is one of the mediators of the remodeling of spiral arteries throughout pregnancy. The D allele of the Insertion/Deletion (I/D) polymorphism in the ACE gene has been associated with higher ACE activity, accounting for 47% of the total phenotypic variance of serum enzyme levels. To investigate whether the ACE I/D polymorphism affects maternal uteroplacental and fetal umbilical circulation and the pregnancy outcome in women with a history of preeclampsia, 106 women underwent Doppler examination of uterine arteries resistance index and umbilical artery pulsatility index at the 16th, 20th, and 24th weeks of gestation and were genotyped for the I/D polymorphism. This study found a difference in genotype distribution (
P
=0.0002) and allele frequency (
P
<0.0001) between women with and those without preeclampsia recurrence and fetal growth restriction as well as an association (
P
=0.0007) between DD genotype and risk of recurrent preeclampsia or fetal growth restriction. At the 16th, 20th, and 24th weeks, uterine artery resistance indexes were significantly lower in II, higher in DD, and intermediate in ID genotype carriers, whereas the umbilical artery pulsatility index values were significantly higher in the DD group in comparison to ID and II genotypes. The current study shows that the ACE I/D polymorphism affects uteroplacental and umbilical flows and the recurrence of an adverse pregnancy outcome in women with history of preeclampsia.
The Role of
Wnk4
in Polygenic Hypertension
Authors: Monti Jan , Zimdahl Heike , Schulz Herbert , Plehm Ralph , Ganten Detlev , Hübner Norbert
Published: April 2003

Linkage analyses in experimental crosses of stroke-prone spontaneously hypertensive (SHRSP) and normotensive Wistar-Kyoto (WKY) rats have strongly suggested the presence of quantitative trait loci (QTL) influencing blood pressure and
ACE
levels on rat chromosome 10, which have been confirmed in multiple independent studies. Analysis of the orthologous region on human chromosome 17 also revealed significant linkage to blood pressure in several populations.
Wnk4
, a gene previously identified to cause pseudohypoaldosteronism type II, a rare mendelian form of arterial hypertension, is located on human chromosome 17. The hypothesis has been advanced that molecular variants of this gene might contribute to common polygenic forms of hypertension, since
Wnk4
is located in a region of conserved synteny that demonstrates an overlap between quantitative trait loci for primary hypertension in humans and rats. In this report, we describe the confirmation of the blood pressure QTL on rat chromosome 10 by congenic approaches, spanning the
Wnk4
locus. Comparative analysis of the complete coding sequence of
Wnk4
in SHRSP and WKY strains revealed no mutation and demonstrated high conservation between rat and human proteins. Furthermore, comparison of mRNA levels in the kidney showed no differences between SHRSP and WKY. Additionally, we excluded a secondary effect of blood pressure on the transcriptional regulation of
Wnk4
. Our results fail to support a material contribution of
Wnk4
to blood pressure regulation in this model of polygenic hypertension. Thus,
Wnk4
is likely not to represent the underlying disease gene for the QTL captured in chromosome 10 congenic animals.
Calcium Antagonist Treatment by Lercanidipine Prevents Hyperpolarization in Essential Hypertension
Authors: Taddei Stefano , Virdis Agostino , Ghiadoni Lorenzo , Versari Daniele , Salvetti Guido , Magagna Armando , Salvetti Antonio
Published: April 2003

Essential hypertension is associated with impaired endothelium-dependent vasodilation caused by oxidative stress-induced nitric oxide (NO) breakdown and compensatory production of a hyperpolarizing factor. To test whether calcium antagonist treatment can restore NO availability and prevent hyperpolarization through antioxidant properties, in 15 healthy subjects and 15 patients with essential hypertension, we studied forearm blood flow (strain-gauge plethysmography) modifications induced by intrabrachial bradykinin (5, 15, 50 ng/100 mL per minute), an endothelium-dependent vasodilator, in basal conditions, during infusion of
N
G
-monomethyl-
l
-arginine (L-NMMA, 100 μg/100 mL per minute), an NO-synthase inhibitor, and ouabain (0.72 μg/100 mL per minute), an Na
+
-K
+
ATPase inhibitor to prevent hyperpolarization. These infusions were repeated in the presence of the antioxidant vitamin C (8 mg/100 mL/min). The response to sodium nitroprusside was also evaluated. In controls, vasodilation to bradykinin was inhibited by L-NMMA and remained unchanged by ouabain or vitamin C. In hypertensive patients, vasodilation to bradykinin was blunted and resistant to L-NMMA but sensitive to ouabain. Vitamin C increased the response to bradykinin and restored the inhibiting effect of L-NMMA while preventing the effect of ouabain. In hypertensive patients, infusions were repeated after 3-month treatment with lercanidipine (10 to 20 mg daily). Lercanidipine decreased plasma lipoperoxides, isoprostanes, and malondialdehyde and increased plasma antioxidant capacity. Moreover, lercanidipine increased the vasodilation to bradykinin and restored the inhibiting effect of L-NMMA on bradykinin-induced vasodilation while preventing the effect of ouabain. Finally, vitamin C no longer exerted its facilitating activity. These results indicate that in essential hypertension, lercanidipine increases endothelium-dependent vasodilation by restoring NO availability and preventing hyperpolarization, an effect probably determined by antioxidant activity.
Coronary Myogenic Constriction Antagonizes EDHF-Mediated Dilation
Authors: Gschwend Simone , Henning Robert H. , de Zeeuw Dick , Buikema Hendrik
Published: April 2003

In hypertension, pressure-induced myogenic constriction and impaired endothelium-derived hyperpolarizing factor (EDHF)-mediated dilation may contribute to increased vasomotor tone. Myogenic constriction as well as EDHF-mediated dilation may share common signaling mechanisms, and both may control K
Ca
channel activity to set arterial tone. To investigate a potential relation between the 2 mechanisms, we studied coronary arteries of Sprague-Dawley rats for individual myogenic constriction compared with EDHF-mediated dilation of the same artery. EDHF-mediated dilation was measured as the maximal dilation to acetylcholine (100 μmol/L) after preconstriction, resistant to NO inhibition (
N
G
-methyl-
l
-arginine acetate salt, L-NMMA, 100 μmol/L), and prostaglandin inhibition (indomethacin, 10 μmol/L) but abolished by charybdotoxin (100 nmol/L) plus apamin (500 nmol/L). Individual coronary myogenic constriction at an intraluminal pressure of 70 mm Hg (n=9) ranged from 6% to 44% (24±4%). EDHF-mediated dilation ranged from 18% to 84% (42±7%). Elevating pressure to 130 mm Hg (n=8) increased myogenic constriction by 2-fold (
P
<0.01) and decreased EDHF-mediated dilation by 2.6-fold (
P
<0.01). Interestingly, individual myogenic constriction inversely correlated to individual EDHF-mediated dilation (
r
=−0.75,
P
<0.001, n=17). Pretreatment with the K
Ca
channel opener NS1619 (30 μmol/L) prevented coronary myogenic constriction and increased EDHF-mediated dilation by 2.2-fold (
P
<0.01), whereas the K
ATP
channel opener cromakalim (3 μmol/L) had no effect on EDHF-mediated dilation. For comparison, in mesenteric arteries (at 70 mm Hg) low myogenic constriction (2±1%) was associated with high EDHF-mediated dilation (93±2%), and pretreatment with NS1619 had no effect. Our results demonstrate that myogenic constriction in coronary arteries antagonizes EDHF-mediated dilation. Activation of K
Ca
channels with NS1619 reduces myogenic constriction and profoundly increases EDHF-mediated dilation, specifically in coronary arteries, suggesting a potential therapeutic impact to reduce coronary risk in hypertension.
Ral GDP Dissociation Stimulator and Ral GTPase Are Involved in Myocardial Hypertrophy
Authors: Kawai Miki , Kawashima Seinosuke , Sakoda Tsuyoshi , Toh Ryuji , Kikuchi Akira , Yamauchi-Takihara Keiko , Kunisada Keita , Yokoyama Mitsuhiro
Published: April 2003

Ras-related GTPase (Ral) is converted to the GTP-bound form by Ral GDP dissociation stimulator (Ral-GDS), a putative effector protein of Ras. Although a number of studies indicate that Ras induces cardiac hypertrophy, the functional role of Ral-GDS/Ral signaling pathway is as yet unknown in cardiac myocytes. We investigated the role of the Ral-GDS/Ral pathway in cardiac hypertrophy. Transfection of Ral-GDS and constitutively active mutant of Ral (RalG23V) in cultured rat neonatal myocytes stimulated promoter activity of c-
fos
(5.4-fold and 2.6-fold,
P
<0.01), α-skeletal actin (2.7-fold and 2.1-fold,
P
<0.01), and β-myosin heavy chain-luciferase (2.8-fold and 2.3-fold,
P
<0.01). Ral-GDS–induced or RalG23V-induced promoter activation was increased synergistically with activated Ras (RasG12V). Dominant-negative mutant of Ral (RalS28N) partially inhibited RasG12V induced promoter activation. Cardiac myocytes transfected with RalG23V showed increased cell size compared with nontransfected or vector-transfected cells (2.1-fold,
P
<0.01). Cardiotrophin-1 (CT-1) upregulated Ral-GDS mRNA expression and induced Ral activation. CT-1–induced Ral-GDS mRNA expression was inhibited by overexpression of the dominant-negative mutant of STAT3. Moreover, Ral activity was elevated in hypertrophied hearts (2.1-fold,
P
<0.01) by mechanical stress in association with increased CT-1 expression and signal transducer and activator of transcription 3 (STAT3) phosphorylation in the rat aortic banding model. Ral-GDS/Ral pathway is involved in a wide range of gene expressions and is activated by hypertrophic stimuli in vitro and in vivo. SATA3 may play a key role in Ral-GDS expression and Ral activation. Our data provide evidence that the Ral-GDS/Ral signaling pathway is a link to the process of cardiac hypertrophy.
Dietary Salt Regulates Renal SGK1 Abundance
Authors: Farjah Mariam , Roxas Bryan P. , Geenen David L. , Danziger Robert S.
Published: April 2003

Serum and glucocorticoid-induced kinase 1 (SGK1) activates the epithelial sodium channel (eNaC) in tubules. We examined renal SGK1 abundance in salt-adaptation and in salt-sensitive hypertension. Sprague-Dawley and Dahl salt-sensitive rats were placed on either 8% or 0.3% NaCl diets for 10 days. Plasma aldosterone levels were ≈2.5-fold greater on 0.3% versus 8% NaCl diets in both rat strains. Both serum and glucocorticoid-induced kinase 1 transcript and protein abundance were less (
P
<0.01) in Sprague-Dawley rats and greater (
P
<0.01) in Dahl salt-sensitive rats on 8% versus 0.3% NaCl diets. The cDNA sequences of serum and glucocorticoid-induced kinase 1 in both strains of rat were the same. The present results provide evidence that the abundance of serum and glucocorticoid-induced kinase 1 in rat kidney may play a role in salt adaptation and the pathogenesis of hypertension and suggests that aldosterone is not the primary inducer of SGK1 in the Sprague-Dawley rat.
Transmural Heterogeneity of Calcium Handling in Canine
Authors: Laurita Kenneth R. , Katra Rodolphe , Wible Barbara , Wan Xiaoping , Koo Michael H.
Published: April 2003

Spatial heterogeneity of the action potential and its influence on arrhythmia vulnerability is known. However, heterogeneity of intracellular calcium handling and, in particular, its effect on the electrophysiological substrate is less clear. Using optical mapping techniques, calcium transients and action potentials were recorded simultaneously from ventricular sites across the transmural wall of the arterially perfused canine left ventricular wedge preparation during steady-state baseline pacing and rapid pacing. During baseline pacing, the decay of intracellular calcium to diastolic levels and calcium transient duration were slower (70%,
P
<0.005) and longer (20%,
P
<0.005), respectively, closer to the endocardial surface compared with the epicardial surface. Tissue samples isolated from the left ventricular wall demonstrate that sarcoplasmic reticulum Ca
2+
ATPase (SERCA2a) expression was significantly less in the subendocardial and midmyocardial layers compared with the subepicardial layer. In contrast, no significant difference in the transmural expression of Na
+
-Ca
2+
exchanger was observed. During rapid pacing, calcium transient alternans and increased levels of diastolic intracellular calcium were significantly greater (
P
<0.01) closer to the endocardium (101%±62% and 41%±15%, respectively) compared with the epicardium (12%±7% and 12%±14%, respectively). In conclusion, cells closer to the endocardium exhibit a slower decay of intracellular calcium compared with cells near the epicardium, which may be due in part to reduced expression of SERCA2a. As a possible consequence, calcium transient alternans and increased diastolic levels of intracellular calcium may occur preferentially closer to the endocardial surface.
Cellular Basis of Abnormal Calcium Transients of Failing Human Ventricular Myocytes
Authors: Piacentino Valentino , Weber Christopher R. , Chen Xiongwen , Weisser-Thomas Jutta , Margulies Kenneth B. , Bers Donald M. , Houser Steven R.
Published: April 2003

Depressed contractility is a central feature of the failing human heart and has been attributed to altered [Ca
2+
]
i
. This study examined the respective roles of the L-type Ca
2+
current (
I
Ca
), SR Ca
2+
uptake, storage and release, Ca
2+
transport via the Na
+
-Ca
2+
exchanger (NCX), and Ca
2+
buffering in the altered Ca
2+
transients of failing human ventricular myocytes. Electrophysiological techniques were used to measure and control V
m
and measure
I
m
, respectively, and Fluo-3 was used to measure [Ca
2+
]
i
in myocytes from nonfailing (NF) and failing (F) human hearts. Ca
2+
transients from F myocytes were significantly smaller and decayed more slowly than those from NF hearts. Ca
2+
uptake rates by the SR and the amount of Ca
2+
stored in the SR were significantly reduced in F myocytes. There were no significant changes in the rate of Ca
2+
removal from F myocytes by the NCX, in the density of NCX current as a function of [Ca
2+
]
i
,
I
Ca
density, or cellular Ca
2+
buffering. However, Ca
2+
influx during the late portions of the action potential seems able to elevate [Ca
2+
]
i
in F but not in NF myocytes. A reduction in the rate of net Ca
2+
uptake by the SR slows the decay of the Ca
2+
transient and reduces SR Ca
2+
stores. This leads to reduced SR Ca
2+
release, which induces additional Ca
2+
influx during the plateau phase of the action potential, further slowing the decay of the Ca
2+
transient. These changes can explain the defective Ca
2+
transients of the failing human ventricular myocyte.
The Antiinflammatory Endothelial Tyrosine Kinase Tie2 Interacts With a Novel Nuclear Factor-κB Inhibitor ABIN-2
Authors: Hughes David P. , Marron Marie B. , Brindle Nicholas P.J.
Published: April 2003

Tie2 is a receptor tyrosine kinase expressed predominantly in endothelial cells and is essential for blood vessel formation and maintenance. The receptor has potent antiinflammatory effects on endothelial cells, suppressing vascular endothelial growth factor– and tumor necrosis factor–induced expression of leukocyte adhesion molecules and procoagulant tissue factor and inhibiting vascular leakage. To delineate the signaling pathways utilized by Tie2, we performed yeast two-hybrid screening of a human endothelial cell cDNA library and identified a novel protein interacting with the intracellular domain of the receptor. This protein was found to be human A20 binding inhibitor of NF-κB activation-2, ABIN-2, an inhibitor of NF-κB–mediated inflammatory gene expression. Coexpression of Tie2 and ABIN-2 in CHO cells confirmed the interaction occurs in mammalian cells. In contrast, Tie1 did not interact with ABIN-2 in the yeast two-hybrid system or mammalian cells. Deletion analysis identified the Tie2 binding motif to be encompassed between residues 171 and 272 in ABIN-2. Interaction was dependent on Tie2 autophosphorylation but ABIN-2 was not tyrosine phosphorylated by Tie2. Furthermore, in endothelial cells the interaction was stimulated by the Tie2 ligand angiopoietin-1. Expression of ABIN-2 deletion mutants in endothelial cells suppressed the ability of angiopoietin-1 to inhibit phorbol ester–stimulated NF-κB–dependent reporter gene activity. These findings provide the first direct link between Tie2 and a key regulator of inflammatory responses in endothelial cells. Interaction between Tie2 and ABIN-2 may be important in the vascular protective antiinflammatory actions of Tie2.
Novel NAD(P)H Oxidase Inhibitor Suppresses Angioplasty-Induced Superoxide and Neointimal Hyperplasia of Rat Carotid Artery
Authors: Jacobson Gary M. , Dourron Hector M. , Liu Jianhua , Carretero Oscar A. , Reddy Daniel J. , Andrzejewski Tanja , Pagano Patrick J.
Published: April 2003

Neointimal proliferation occurring after vascular or endovascular procedures is a major complication leading to end-organ or limb ischemia. In experimental models, balloon injury has been shown to induce NAD(P)H oxidase to produce vascular superoxide anion (O
2
·−
) production, which has been implicated in cell proliferation, but a direct link is still unclear. We postulated that inhibition of arterial NAD(P)H oxidase, resulting in decreased O
2
·−
, would lessen the neointimal hyperplasia caused by balloon injury to the common carotid artery (CCA). Sprague-Dawley rats were implanted with osmotic minipumps containing either vehicle, a cell-permeant peptide that inhibits NAD(P)H oxidase (gp91ds-tat, 10 mg/kg per day), or a scrambled peptide control (scrmb-tat). Two days after pump implantation, the left CCA was injured using an intravascular balloon embolectomy catheter (2F Fogarty). Systolic blood pressure was monitored by tail cuff. Fourteen days after injury, CCAs were harvested and analyzed by digital morphometry. Rats in both groups remained normotensive, with no significant differences in systolic blood pressure. Reactive oxygen species measurements after injury indicated a significant reduction in vascular O
2
·−
in rats infused with gp91ds-tat, and the neointima/media area and thickness ratios were significantly lower in their arteries compared with control. On the contrary, no significant change in overall CCA diameter was observed in any group. Our data indicate that in response to balloon injury of the rat carotid artery, NAD(P)H oxidase activity contributes to neointimal hyperplasia and is involved in vascular cell proliferation and migration during restenosis.
Increased Superoxide Generation Is Associated With Pulmonary Hypertension in Fetal Lambs
Authors: Brennan Lisa A. , Steinhorn Robin H. , Wedgwood Stephen , Mata-Greenwood Eugenia , Roark Everett A. , Russell James A. , Black Stephen M.
Published: April 2003

Ligation of the ductus arteriosus in utero produces pulmonary hypertension and vascular remodeling in fetal and newborn lambs. However, the mechanisms producing these vascular changes are not well defined. Because reactive oxygen species (ROS) have been implicated as mediators of smooth muscle cell proliferation, we hypothesized that increased formation of ROS may be involved in the pathophysiology of pulmonary hypertension after in utero ductal ligation. Using ethidium fluorescence, we demonstrated an increase in superoxide levels after 9 days of ductal ligation compared with control lungs (
P
<0.05) that was localized to the adventitia and smooth muscle cells of hypertensive vessels. SOD-1 and SOD-2 protein levels and activities in lung, vein, and artery of hypertensive lambs were unchanged relative to controls after 2 days of ductal ligation. However, after 9 days, superoxide dismutase (SOD) activity was significantly decreased in arteries from ligated lambs without associated changes in SOD protein expression (
P
<0.05). Examination of NADPH oxidase expression as a potential source of the superoxide production indicated that the levels of p67
phox
, a subunit of the NADPH oxidase complex, were significantly increased in the pulmonary arteries, but not veins, from the ligated lung as early as 2 days (
P
<0.05). Functional analyses demonstrated that reducing superoxide levels significantly increased the NO-mediated relaxation of pulmonary arteries isolated after 9 days, but not 2 days, of ductal ligation (
P
<0.05). These results suggest that increased NADPH oxidase expression may increase levels of superoxide in persistent pulmonary hypertension of the newborn lung tissue, and that increased superoxide blunts vascular relaxations to exogenous NO while stimulating smooth muscle cell growth.
Intravenous Administration of Human Bone Marrow Stromal Cells Induces Angiogenesis in the Ischemic Boundary Zone After Stroke in Rats
Authors: Chen Jieli , Zhang Zheng Gang , Li Yi , Wang Lei , Xu Yong Xian , Gautam Subhash C. , Lu Mei , Zhu Zhenping , Chopp Michael
Published: April 2003

We tested the hypothesis that intravenous infusion of human bone marrow stromal cells (hMSCs) promotes vascular endothelial growth factor (VEGF) secretion, VEGF receptor 2 (VEGFR2) expression and angiogenesis in the ischemic boundary zone (IBZ) after stroke. hMSCs (1×10
6
) were intravenously injected into rats 24 hours after middle cerebral artery occlusion (MCAo). Laser scanning confocal microscopy (LSCM), immunohistochemistry and ELISA were performed to assay angiogenesis and levels of human and rat VEGF in the host brain, respectively. In addition, capillary-like tube formation was measured using mouse brain-derived endothelial cells (MBDECs). Morphological and three dimensional image analyses revealed significant (
P
<0.05) increases in numbers of enlarged and thin walled blood vessels and numbers of newly formed capillaries at the boundary of the ischemic lesion in rats (n=12) treated with hMSCs compared with numbers in rats (n=12) treated with PBS. ELISA measurements showed that treatment with hMSCs significantly (
P
<0.05) raised endogenous rat VEGF levels in the IBZ from 10.5±1.7 ng/mL in the control group to 17.5±1.6 ng/mL in the hMSC-treated group. In addition, treatment with hMSCs increased endogenous VEGFR2 immunoreactivity. In vitro, when MBDECs were incubated with the supernatant obtained from cultured hMSCs, capillary-like tube formation was significantly (
P
<0.01) induced. However, hMSC-induced capillary-like tube formation was significantly (
P
<0.01) inhibited when the endothelial cells were incubated with the supernatant from hMSCs in the presence of a neutralizing anti-VEGFR2. These data suggest that treatment of stroke with hMSCs enhances angiogenesis in the host brain and hMSC-enhanced angiogenesis is mediated by increases in levels of endogenous rat VEGF and VEGFR2.
Endothelial Cells Secrete Triglyceride Lipase and Phospholipase Activities in Response to Cytokines as a Result of Endothelial Lipase
Authors: Jin Weijun , Sun Gwo-Shing , Marchadier Dawn , Octtaviani Edelyn , Glick Jane M. , Rader Daniel J.
Published: April 2003

The endothelium interacts extensively with lipids and lipoproteins, but there are very few data regarding the ability of endothelial cells to secrete lipases. In this study, we investigated the ability of endothelial cells to secrete the triglyceride lipase and phospholipase activities characteristic of endothelial lipase (EL), a recently described member of the triglyceride lipase gene family. No lipase activities were detected under basal conditions, but treatment with cytokines significantly stimulated the expression of both activities. Using antibodies to EL, we determined that both activities were primarily a result of this enzyme. In addition to the increase in lipolytic activity, cytokine treatment was demonstrated to substantially upregulate EL protein and EL mRNA in a dose-dependent manner. Cytokines did not change EL mRNA stability. Both new protein synthesis and activation of NF-κB influenced the induction of EL by cytokines, suggesting that multiple pathways contribute to this process. The upregulation of EL by cytokines is in sharp contrast to the downregulation by cytokines of the other two major members of this gene family, lipoprotein lipase and hepatic lipase, and has implications for the physiological role of EL in inflammatory conditions and its potential role in the modulation of lipoprotein metabolism during inflammatory conditions, including atherosclerosis.
Calmodulin Regulation of Excitation-Contraction Coupling in Cardiac Myocytes
Authors: Yang Dongmei , Song Long-Sheng , Zhu Wei-Zhong , Chakir Khalid , Wang Wei , Wu Caihong , Wang Yibin , Xiao Rui-Ping , Chen S.R. Wayne , Cheng Heping
Published: April 2003

Calmodulin (CaM) as a ubiquitous Ca
2+
sensor interacts with multiple key molecules involved in excitation-contraction (EC) coupling. In the present study, we report that adenoviral expression of a mutant CaM lacking all of its four Ca
2+
-binding sites, CaM(1-4), at a level 6.5-fold over endogenous CaM markedly increases the amplitude and abbreviates the decay time of Ca
2+
transients and contraction in cultured rat ventricular myocytes. To determine the underlying mechanisms, we examined the properties of L-type Ca
2+
channels, Ca
2+
/CaM-dependent protein kinase II (CaMKII), and phospholamban (PLB) in the sarcoplasmic reticulum (SR). We found that CaM(1-4) expression markedly augmented L-type Ca
2+
current amplitude and slowed its inactivation. Surprisingly, overexpression of CaM(1-4) increased CaMKII activity and phosphorylation of PLB-Thr-17. Moreover, CaM(1-4) elevated diastolic Ca
2+
and caffeine-labile Ca
2+
content of the SR. Inhibition of CaMKII by KN-93 or a myristoylated autocamtide-2 related inhibitory peptide prevented the aforementioned PLB phosphorylation and reversed the positive inotropic and relaxant effects, indicating that CaMKII is essential to CaM(1-4) actions. These results demonstrate that CaM modulates Ca
2+
influx, SR Ca
2+
release, and Ca
2+
recycling during cardiac EC coupling. A novel finding of this study is that expression of a Ca
2+
-insensitive CaM mutant can lead to activation of CaMKII in cardiac myocytes.
12-Lipoxygenase in Opioid-Induced Delayed Cardioprotection
Authors: Patel Hemal H. , Fryer Ryan M. , Gross Eric R. , Bundey Richard A. , Hsu Anna K. , Isbell Marilyn , Eusebi Leonard O.V. , Jensen Roderick V. , Gullans Steven R. , Insel Paul A. , Nithipatikom Kasem , Gross Garrett J.
Published: April 2003

12-Lipoxygenase (12-LO) has been shown to be a factor in acute ischemic preconditioning (IPC) in the isolated rat heart; however, no studies have been reported in delayed PC. We characterized the role of 12-LO in an intact rat model of delayed PC induced by a δ-opioid agonist SNC-121 (SNC). Rats were pretreated with SNC and allowed to recover for 24 hours. They were then treated with either baicalein or phenidone, 2 selective 12-LO inhibitors. In addition, SNC-pretreated rats had plasma samples isolated at different times after ischemia-reperfusion for liquid chromatographic–mass spectrometric analysis of the major metabolic product of 12-LO, 12-HETE. Similar studies were conducted with inhibitors. Gene array data showed a significant induction of 12-LO message (
P
<0.05) after opioid pretreatment. This induction in 12-LO mRNA was confirmed by real-time polymerase chain reaction, and 12-LO protein expression was enhanced by SNC pretreatment at 24 hours relative to vehicle treatment. Both baicalein and phenidone attenuated the protective effects of SNC pretreatment on infarct size (50±4% and 42±3% versus 29±2%,
P
<0.05, respectively). No significant differences were observed in 12-HETE concentrations between baseline control and SNC-treated rats. However, 12-HETE concentrations were increased significantly at both 15 minutes during ischemia and at 1 hour of reperfusion in the SNC-treated rats compared with controls. Baicalein and phenidone attenuated the increase in 12-HETE at 1 hour of reperfusion. These data suggest that SNC-121 appears to enhance message and subsequently the activity and expression of 12-LO protein during times of stress, resulting in delayed cardioprotection.
Protein Kinase C Signaling Transduces Endorphin-Primed Cardiogenesis in GTR1 Embryonic Stem Cells
Authors: Ventura Carlo , Zinellu Elisabetta , Maninchedda Emiliana , Fadda Marina , Maioli Margherita
Published: April 2003

The prodynorphin gene and its product, dynorphin B, have been found to promote cardiogenesis in embryonic cells by inducing the expression of GATA-4 and Nkx-2.5, two transcription factor–encoding genes essential for cardiogenesis. The molecular mechanism(s) underlying endorphin-induced cardiogenesis remain unknown. In the present study, we found that GTR1 embryonic stem (ES) cells expressed cell surface κ opioid receptors, as well as protein kinase C (PKC)-α, -β
1
, -β
2
, -δ, -ε, and -ζ. Cardiac differentiation was associated with a marked increase in the B
max
value for a selective opioid receptor ligand and complex subcellular redistribution of selected PKC isozymes. PKC-α, -β
1
, -β
2
, -δ, and -ε all increased in the nucleus of ES-derived cardiac myocytes, compared with nuclei from undifferentiated cells. In both groups of cells, PKC-δ and -ε were mainly expressed at the nuclear level. The nuclear increase of PKC-α, -β
1
, and -β
2
was due to a translocation from the cytosolic compartment. In contrast, the increase of both PKC-δ and PKC-ε in the nucleus of ES-derived cardiomyocytes occurred independently of enzyme translocation, suggesting changes in isozyme turnover and/or gene expression during cardiogenesis. No change in PKC-ζ expression was observed during cardiac differentiation. Opioid receptor antagonists prevented the nuclear increase of PKC-α, PKC-β
1
, and PKC-β
2
and reduced cardiomyocyte yield but failed to affect the nuclear increase in PKC-δ and -ε. PKC inhibitors prevented the expression of cardiogenic genes and dynorphin B in ES cells and abolished their development into beating cardiomyocytes.
Dynorphin B Is an Agonist of Nuclear Opioid Receptors Coupling Nuclear Protein Kinase C Activation to the Transcription of Cardiogenic Genes in GTR1 Embryonic Stem Cells
Authors: Ventura Carlo , Zinellu Elisabetta , Maninchedda Emiliana , Maioli Margherita
Published: April 2003

The cardiac differentiation of embryonic stem (ES) cells was found to involve prodynorphin gene and dynorphin B expression and was associated with the interaction of secreted dynorphin B with cell surface opioid receptors coupled with protein kinase C (PKC) signaling and complex subcellular redistribution patterning of selected PKC isozymes. Here, confocal microscopy revealed the presence of immunoreactive dynorphin B–like material in GTR1 ES cells, suggesting that dynorphin peptides may also act intracellularly. Opioid binding sites were identified in ES cell nuclei, with a single dissociation constant in the low nanomolar range. A significant increase in B
max
for a κ opioid receptor ligand was observed in nuclei isolated from ES-derived cardiomyocytes compared with nuclei from undifferentiated cells. Direct exposure of nuclei isolated from undifferentiated ES cells to dynorphin B or U-50,488H, a synthetic κ opioid receptor agonist, time- and dose-dependently activated the transcription of GATA-4 and Nkx-2.5, 2 cardiac lineage–promoting genes. Nuclear exposure to dynorphin B also enhanced the rate of prodynorphin gene transcription. These responses were abolished in a stereospecific fashion by the incubation of isolated nuclei with selective opioid receptor antagonists. Nuclei isolated from undifferentiated cells were able to phosphorylate the acrylodan-labeled MARCKS peptide, a high-affinity fluorescent PKC substrate. Exposure of isolated nuclei to dynorphin B induced a remarkable increase in nuclear PKC activity, which was suppressed by opioid receptor antagonists. Nuclear treatment with PKC inhibitors abolished the capability of dynorphin B to prime the transcription of cardiogenic genes.
Reengineering Inducible Cardiac-Specific Transgenesis With an Attenuated Myosin Heavy Chain Promoter
Authors: Sanbe Atsushi , Gulick James , Hanks Mark C. , Liang Qiangrong , Osinska Hanna , Robbins Jeffrey
Published: April 2003

Despite the advantages of reversibly altering cardiac transgene expression, the number of successful studies with inducible cardiac-specific transgene expression remains limited. The utility of the current system is hampered by the large number of lines needed before a nonleaky inducible line is isolated and by the use of a heterologous virus-based minimal promoter in the responder line. We developed an efficient, experimentally flexible system that enables us to reversibly affect both abundant and nonabundant cardiomyocyte proteins. The use of bacterial-codon–based transactivators led to aberrant splicing, whereas other more efficient transactivators, by themselves, caused disease when expressed in the heart. The redesign of the system focused on developing stable transactivator-expressing lines in which expression was driven by the mouse α-myosin heavy chain promoter. A minimal responder locus was derived from the same promoter, in which the GATA sites and thyroid responsive elements responsible for robust cardiac specific expression were ablated, leading to an attenuated promoter that could be inducibly controlled. In all cases, whether activated or not, expression mimicked that of the parental promoter. By use of this system, an inducible expression of an abundant contractile protein, the atrial isoform of essential myosin light chain 1, and a powerful biological effector, glycogen synthase kinase-3β (GSK-3β), were obtained. Subsequently, we tested the hypothesis that GSK-3β expression could reverse a preexisting hypertrophy. Inducible expression of GSK-3β could both attenuate a hypertrophic response and partially reverse a pressure-overload–induced hypertrophy. The system appears to be robust and can be used to temporally control high levels of cardiac-specific transgene expression.
Neural Stem Cells
Authors: Galli Rossella , Gritti Angela , Bonfanti Luca , Vescovi Angelo Luigi
Published: April 2003

This review focuses on the nature and functional properties of stem cells of the adult mammalian central nervous system (CNS). It has recently been shown that cell turnover, including neurons, does occur in the mature CNS, thanks to the persistence of precursor cells that possess the functional characteristics of bona-fide neural stem cells (NSCs) within restricted brain areas. We discuss how the subventricular zone of the forebrain (SVZ) is the most active neurogenetic area and the richest source of NSCs. These NSCs ensure a life-long contribution of new neurons to the olfactory bulb and, when placed in culture, can be grown and extensively expanded for months, allowing the generation of stem cell lines, which maintain stable and constant functional properties. A survey of the differentiation potential of these NSCs, both in vitro and in vivo, outlines their extreme plasticity that seems to outstretch the brain boundaries, so that these neuroectodermal stem cells may give rise to cells that derive from developmentally distinct tissues. A critical discussion of the latest, controversial findings regarding this surprising phenomenon is provided.
The Endothelial Glycocalyx Protects Against Myocardial Edema
Authors: van den Berg Bernard M. , Vink Hans , Spaan Jos A.E.
Published: April 2003

Myocardial tissue edema attributable to increased microvascular fluid loss contributes to cardiac dysfunction after myocardial ischemia, cardiopulmonary bypass, hypertension, and sepsis. Recent studies suggest that carbohydrate structures on the luminal surface of microvascular endothelium are essential to prevent tissue edema. We carefully preserved these structures for visualization with electron microscopy, revealing that the rat myocardial capillary endothelial surface is coated with a 0.2- to 0.5-μm-thick carbohydrate layer and that its degradation instantly results in notable myocardial tissue edema.
Factor Associated With Neutral Sphingomyelinase Activation and Its Role in Cardiac Cell Death
Authors: O’Brien Nicole W. , Gellings Nicole M. , Guo Mei , Barlow Steven B. , Glembotski Christopher C. , Sabbadini Roger A.
Published: April 2003

Generation of proapoptotic sphingolipids by neutral sphingomyelinase activation is an early response to hypoxia/reoxygenation (HR) in cardiomyocytes.
F
actor
a
ssociated with
n
eutral sphingomyelinase activation (FAN) mediates activation of sphingomyelinase and subsequent apoptosis. However, the participation of FAN in HR-induced cardiomyocyte cell death has not been elucidated. We therefore investigated the expression and role of FAN in rat cardiomyocytes. A cDNA was isolated from rat heart encoding putative rat FAN. Reverse transcriptase–polymerase chain reaction, immunoelectron microscopy, and immunofluorescence demonstrated for the first time the expression of FAN specifically in rat cardiomyocytes. FAN expression was confirmed by the finding that expression of a dominant-negative FAN almost completely abrogated HR-induced cell death, whereas overexpression of wild-type FAN led to an increase. Treatment of FAN and dominant-negative FAN–expressing cells with C2-ceramide produced substantial cell death, indicating dominant-negative FAN exerts its protective action by interfering with the activation of the sphingolipid cascade. Taking these results together, we conclude that FAN is a previously undescribed and important HR signaling component in the heart and that inhibition of FAN may provide a novel intervention point for reducing ischemia/reperfusion injury.
Sildenafil Induces Delayed Preconditioning Through Inducible Nitric Oxide Synthase–Dependent Pathway in Mouse Heart
Authors: Salloum Fadi , Yin Chang , Xi Lei , Kukreja Rakesh C.
Published: April 2003

Sildenafil citrate (Viagra) is the most widely used drug for treating erectile dysfunction in men. We recently demonstrated that it induces potent protective effects against ischemia-reperfusion (I-R) injury in rabbit hearts through the opening of mitochondrial ATP-dependent K
+
channels. In the present study, we investigated the role of the NO-dependent signaling pathway in delayed cardioprotection by sildenafil. Adult male ICR mice were treated with saline or sildenafil (0.7 mg/kg IP) 24 hours before global I-R in the Langendorff mode. Infarct size was reduced from 27.6±3.3% in saline-treated control mice to 6.9±1.2% in sildenafil-treated mice (mean±SEM,
P
<0.05) without compromising cardiac function. Reverse transcription–polymerase chain reaction revealed a transient increase in endothelial and inducible NO synthase (eNOS and iNOS, respectively) mRNA in sildenafil-treated mice, peaking at 45 minutes (eNOS) and 2 hours (iNOS) after sildenafil injection. The magnitude of mRNA increase was more pronounced for iNOS than for eNOS. In addition, a significant increase in both iNOS and eNOS protein was detected 24 hours after sildenafil treatment. A selective inhibitor of iNOS, 1400W (10 mg/kg IP given 30 minutes before I-R), abolished sildenafil-induced protection (23.7±2.8%,
P
<0.05 versus sildenafil). These data suggest that the induction of NO synthase isoforms is an essential component of the signaling mechanism for sildenafil-induced delayed preconditioning. However, iNOS appears to be the primary isoform that mediates the robust cardioprotection.
Effects of Race and Poverty on the Process and Outcome of Inpatient Rehabilitation Services Among Stroke Patients
Authors: Horner Ronnie D. , Swanson Jeffrey W. , Bosworth Hayden B. , Matchar David B.
Published: April 2003

Background and Purpose—

The greater mortality and residual physical impairments among black stroke patients may be attributable to differential utilization of rehabilitation services. This report examines, within an equal-access healthcare system, racial differences in time to initiation of stroke rehabilitation services and in the trajectory of physical function recovery.

Methods—

This study was a secondary analysis of data from an inception cohort of 1073 stroke patients hospitalized between April 1995 and March 1997 and followed up for up to 1 year. Inpatient data came from medical record reviews; follow-up data came from telephone interviews at 1, 6, and 12 months after stroke. The study included consecutive acute ischemic or intracerebral hemorrhagic stroke patients from 9 VA medical centers. The main outcome measures were time to initiation of inpatient rehabilitation services and ability to perform activities of daily living.

Results—

There were no racial differences in receipt of inpatient rehabilitation services (blacks, 76%; whites, 70%) or in the proportion of patients referred within 3 days of admission (blacks, 43.5%; whites, 42.0%). Among patients who experienced delay in initiation of rehabilitation, only low-income blacks experienced worse functional recovery over 12 months.

Conclusions—

Low-income black stroke patients who experience delay in initiation of inpatient rehabilitation have a worse trajectory of functional recovery in the first year after stroke. Poverty-associated factors in the postdischarge setting may explain this phenomenon.
Fibrinogen Gene Promoter −455 A Allele as a Risk Factor for Lacunar Stroke
Authors: Martiskainen M. , Pohjasvaara T. , Mikkelsson J. , Mäntylä R. , Kunnas T. , Laippala P. , Ilveskoski E. , Kaste M. , Karhunen P.J. , Erkinjuntti T.
Published: April 2003

Background and Purpose—

Elevated fibrinogen levels are suggested to increase the risk of myocardial infarction and stroke. Carriers of the A allele of the fibrinogen −455G/A polymorphism have increased plasma fibrinogen levels. We studied the association of this polymorphism with stroke subtype in the Stroke Aging Memory (SAM) cohort.

Methods—

The SAM cohort comprises 486 consecutive patients 55 to 85 years of age who, 3 months after ischemic stroke, completed a detailed stroke assessment. Stroke subtypes were examined with MRI. −455G/A genotype was determined by polymerase chain reaction. MRI and genotype data were available for the 299 patients who constitute the present study population.

Results—

Genotype distributions were 64.9% (GG), 31.8% (GA), and 3.3% (AA). In a logistic regression model with age, sex, hypertension, diabetes, hypercholesterolemia, hypertriglyceridemia, myocardial infarction, arrhythmia, atrial fibrillation, peripheral arterial disease, and smoking as possible confounders, there was a significant association between A+ genotype and ≥3 lacunar infarcts (odds ratio [OR], 2.57; 95% CI, 1.23 to 5.36;
P
=0.01). Hypertensive patients carrying the A allele had increased risk (OR, 4.24; 95% CI, 1.29 to 13.99;
P
=0.02) for ≥3 lacunar infarcts. A similar increase in risk was observed among smokers with the A+ genotype (OR, 2.67; 95% CI, 0.92 to 7.77;
P
=0.07).

Conclusions—

Stroke patients carrying the A allele of the Bβ-fibrinogen −455G/A polymorphism frequently presented with multiple lacunar infarcts. This association was stronger among hypertensives and smokers. These associations suggest that the A allele may predispose to atherothrombotic events in cerebrovascular circulation.
Hyperhomocysteinemia Is Associated With the Presence of Left Atrial Thrombus in Stroke Patients With Nonvalvular Atrial Fibrillation
Authors: Ay Hakan , Arsava E. Murat , Tokgözoğlu S. Lale , Özer Necla , Sarıbaş Okay
Published: April 2003

Background and Purpose—

Blood stasis is the fundamental mechanism leading to thrombus formation in the venous system. Homocysteine also poses a significant risk for venous thrombosis through its endothelial toxic and prothrombotic properties. In the present study, we hypothesized that high homocysteine might be associated with thrombus formation in another stasis-related condition, atrial fibrillation.

Methods—

Forty-two consecutive patients with ischemic stroke caused by nonvalvular atrial fibrillation and admitted within the first day of symptom onset were included. Total fasting plasma homocysteine, serum folic acid, and vitamin B12 levels were measured. All patients were evaluated by transesophageal echocardiography for the presence of a left atrial (LA) thrombus. Homocysteine and vitamin levels were compared between groups with or without LA thrombus.

Results—

Transesophageal echocardiography revealed LA thrombus in 20 patients. Mean homocysteine levels were significantly higher in patients with LA thrombus (20.75 versus 13.34 μmol/L,
P
<0.001). Multivariate logistic regression analysis showed that the effect of high homocysteine was independent of other clinical or echocardiographic variables known to increase LA thrombus (
P
=0.017). There was no difference in vitamin B12 levels between groups (
P
=0.118), whereas the mean folic acid level was significantly lower in patients with LA thrombus (
P
=0.004).

Conclusions—

High plasma homocysteine conveys an independent risk for LA thrombus formation in patients with stroke caused by nonvalvular atrial fibrillation. This finding further supports the thrombogenic role of high homocysteine in conditions associated with blood stasis.
High-Density Lipoprotein Cholesterol and Risk of Stroke in Japanese Men and Women
Authors: Soyama Yoshiyuki , Miura Katsuyuki , Morikawa Yuko , Nishijo Muneko , Nakanishi Yumiko , Naruse Yuchi , Kagamimori Sadanobu , Nakagawa Hideaki
Published: April 2003

Background and Purpose—

Evidence of an inverse relationship between serum high-density lipoprotein cholesterol (HDL-C) and the risk of stroke is sparse in Asians and in women. The purpose of this investigation was to examine the relationship in a long-term cohort study of Japanese men and women among whom stroke occurrence is higher than in Western countries.

Methods—

A prospective cohort study was performed involving 4989 participants (1523 men, 3466 women) 35 to 79 years of age at baseline with ≈10 years of follow-up in a rural area of Japan. End points included all stroke incidence and ischemic stroke incidence.

Results—

During follow-up, 132 participants developed stroke, including 81 ischemic stroke cases. Age-adjusted incidence rates per 10 000 person-years for all stroke in subjects with low HDL-C (<30 mg/dL [0.78 mmol/L]) were 103.4 in men and 49.3 in women, which were remarkably higher than in subjects with high HDL-C (≥60 mg/dL [1.56 mmol/L]) (26.4 in men and 15.5 in women). A similar relationship was observed for ischemic stroke. Multivariate-adjusted relative risks for all stroke incidence and ischemic stroke incidence were 2.89 (95% CI, 1.35 to 6.20) and 2.92 (95% CI, 1.17 to 7.32), respectively, for low versus high HDL-C participants. The relationships were independent of sex, age, body mass index, blood pressure, serum total cholesterol, alcohol consumption, and smoking.

Conclusions—

This 10-year follow-up study of Japanese men and women demonstrated that lower HDL-C levels were related significantly and independently to increased risk of all stroke incidence and ischemic stroke incidence.