bluuebunny/crossref_metadata_2025 huggingface dataset:
Abciximab Attenuates Coronary Microvascular Endothelial Dysfunction After Coronary Stenting
Authors: Aymong Eve D. , Curtis Michael J. , Youssef Mostafa , Graham Michelle M. , Shewchuk Lana , Leschuk Wendy , Anderson Todd J.
Published: July 2002

Background

—

Platelet glycoprotein IIb/IIIa receptor blockade with abciximab decreases ischemic events after percutaneous coronary intervention (PCI); however, the mechanism of this benefit has not been fully elucidated. The present study was designed to assess endothelium-dependent vasomotion after coronary stenting and to determine if abciximab alters this response.

Methods and Results

—

The study group consisted of 48 patients (59±10 years of age) with discrete coronary stenoses who underwent stenting alone (n=28) or stenting plus abciximab (n=20). A control group consisted of 31 additional patients who had vasomotor testing on a non-PCI vessel. Coronary blood flow (CBF) was measured (0.014-inch Doppler wire) 30 minutes after uncomplicated PCI and in response to the intracoronary infusion of acetylcholine (Ach) (10
−7
, 10
−6
mol/L Ach) and adenosine (24 μg). Ach-mediated increase in CBF was impaired after stent insertion when compared with the control group (41±52% versus 70±48%;
P
<0.05). The stenting plus abciximab group demonstrated a superior CBF response to Ach compared with the stenting alone group (83±93% versus 41±52%;
P
<0.05), with no difference between groups in the peak flow or percent change in flow to adenosine. By multivariate analysis, concomitant administration of abciximab was strongly predictive of the change in CBF to Ach (
P
<0.005).

Conclusions

—

Abciximab preserves the CBF response to Ach after coronary stenting. The preservation of microvascular endothelial function may help explain the beneficial clinical effect of this agent in patients undergoing PCI.
Clinical and Molecular Characterization of Patients With Catecholaminergic Polymorphic Ventricular Tachycardia
Authors: Priori Silvia G. , Napolitano Carlo , Memmi Mirella , Colombi Barbara , Drago Fabrizio , Gasparini Maurizio , DeSimone Luciano , Coltorti Fernando , Bloise Raffaella , Keegan Roberto , Cruz Filho Fernando E.S. , Vignati Gabriele , Benatar Abraham , DeLogu Angelica
Published: July 2002

Background

—

Mutations in the cardiac ryanodine receptor gene (
RyR2
) underlie catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited arrhythmogenic disease occurring in the structurally intact heart. The proportion of patients with CPVT carrying
RyR2
mutations is unknown, and the clinical features of
RyR2
-CPVT as compared with nongenotyped CPVT are undefined.

Methods and Results

—

Patients with documented polymorphic ventricular arrhythmias occurring during physical or emotional stress with a normal heart entered the study. The clinical phenotype of the 30 probands and of 118 family members was evaluated, and mutation screening on the
RyR2
gene was performed. Arrhythmias documented in probands were: 14 of 30 bidirectional ventricular tachycardia, 12 of 30 polymorphic ventricular tachycardia, and 4 of 30 catecholaminergic idiopathic ventricular fibrillation;
RyR2
mutations were identified in 14 of 30 probands (36% bidirectional ventricular tachycardia, 58% polymorphic ventricular tachycardia, 50% catecholaminergic idiopathic ventricular fibrillation) and in 9 family members (4 silent gene carriers). Genotype-phenotype analysis showed that patients with
RyR2
CPVT have events at a younger age than do patients with nongenotyped CPVT and that male sex is a risk factor for syncope in
RyR2
-CPVT (relative risk=4.2).

Conclusions

—

CPVT is a clinically and genetically heterogeneous disease manifesting beyond pediatric age with a spectrum of polymorphic arrhythmias. β-Blockers reduce arrhythmias, but in 30% of patients an implantable defibrillator may be required. Genetic analysis identifies two groups of patients: Patients with nongenotyped CPVT are predominantly women and become symptomatic later in life; patients with
RyR2
CPVT become symptomatic earlier, and men are at higher risk of cardiac events. These data provide a rationale for prompt evaluation and treatment of young men with
RyR2
mutations.
Myocardial Gene Transfer and Overexpression of β
2
-Adrenergic Receptors Potentiates the Functional Recovery of Unloaded Failing Hearts
Authors: Tevaearai Hendrik T. , Eckhart Andrea D. , Walton G. Brant , Keys Janelle R. , Wilson Katrina , Koch Walter J.
Published: July 2002

Background

—

Mechanical assistance of the failing left ventricle (LV) can lead to functional recovery after a period of unloading, including restoration of β-adrenergic receptor (βAR) inotropic reserve. We tested whether prolonged LV unloading of failing rabbit hearts by use of a heterotopic transplantation technique could lead to recovery and whether adenoviral gene transfer of a β
2
AR transgene (Adv-β
2
AR) could alter this process.

Methods and Results

—

Heart failure was induced by coronary artery ligation in adult New Zealand White rabbits. After 4 weeks, failing hearts were heterotopically transplanted into recipient rabbits, allowing normal coronary perfusion but complete LV unloading. We also placed an LV latex balloon for remote access and in vivo physiological analysis. We found that there was reversal of signaling and functional abnormalities after 30 days of unloading. In another set of failing hearts, we randomly delivered, at the time of transplantation, either 2×10
11
viral particles of Adv-β
2
AR or saline via the coronary arteries. Sham-operated animals with nonfailing hearts served as controls. After 5 days of unloading, in vivo LV contractility (LV dP/dt
max
) and relaxation (LV dP/dt
min
) were significantly decreased in saline-treated failing hearts compared with control nonfailing hearts (
P
<0.05). In failing hearts treated with Adv-β
2
AR, however, LV dP/dt
max
and LV dP/dt
min
were improved in response to higher preloads (
P
<0.05) and βAR stimulation (
P
<0.01).

Conclusions

—

Heterotopic transplantation in the rabbit does allow recovery of the failing heart, and β
2
AR overexpression acutely enhances this functional improvement. Accordingly, genetic manipulation of βAR signaling may represent a novel molecular adjunct to mechanical assistance to facilitate functional myocardial recovery.
Mild Renal Insufficiency Is Associated With Angiographic Coronary Artery Disease in Women
Authors: Reis Steven E. , Olson Marian B. , Fried Linda , Reeser Virginia , Mankad Sunil , Pepine Carl J. , Kerensky Richard , Merz C. Noel Bairey , Sharaf Barry L. , Sopko George , Rogers William J. , Holubkov Richard
Published: July 2002

Background
—

Mild renal insufficiency is associated with an increased risk for cardiovascular events in women with coronary artery disease (CAD). However, the relationship between mild renal insufficiency and atherosclerotic CAD in women is not known.

Methods and Results
—

Women with chest pain who were referred for coronary angiography in the NHLBI Women’s Ischemia Syndrome Evaluation (WISE) study underwent quantitative coronary angiography, blood measurements of creatinine, lipids, and homocysteine, and assessment of CAD risk factors. Fifty-six women had mild renal insufficiency (serum creatinine 1.2 to 1.9 mg/dL), and 728 had normal renal function (creatinine <1.2 mg/dL). Creatinine correlated with angiographic CAD severity score (
r
=0.11,
P
<0.004) and maximum coronary artery stenosis (
r
=0.11,
P
<0.003). Compared with women with normal renal function, those with mild renal insufficiency were more likely to have significant angiographic CAD (≥50% diameter stenosis in ≥1 coronary artery) (61% versus 37%;
P
<0.001) and CAD in multiple vessels (
P
<0.001 for association) and had greater maximum percent diameter coronary stenosis (59±35% versus 38±36%;
P
<0.001). Mild renal insufficiency was associated with significant angiographic CAD independent of age and risk factors (OR=1.9, 95%CI=1.1 to 3.5). After controlling for homocysteine in 509 women, mild renal insufficiency remained predictive of CAD (OR=3.2, 95%CI=1.4 to 7.2).

Conclusions
—

In women with chest pain, mild renal insufficiency is an independent predictor of significant angiographic CAD. Mildly increased serum creatinine is probably a marker for unmeasured proatherogenic factors.
Platelet Nitric Oxide and Superoxide Release During the Development of Nitrate Tolerance
Authors: McVeigh Gary E. , Hamilton Paul , Wilson Martin , Hanratty Colm G. , Leahey William J. , Devine Adrian B. , Morgan David G. , Dixon Lana J. , McGrath Lawrence T.
Published: July 2002

Background

—

The therapeutic benefits that accompany the continuous administration of organic nitrates are attenuated by the development of tolerance to the compounds. Altered superoxide production and NO bioavailability have been implicated in contributing to the development of tolerance, an effect that may be ameliorated by the administration of antioxidants.

Methods and Results

—

We studied the effect of 3 days of continuous transdermal administration of nitroglycerin (NTG) (10 mg/24 hours) on platelet free radical (NO and superoxide anion [O
2
·−
] activity) with and without coadministration of supplemental ascorbate (2.4 g/24 hours). NAD(P)H oxidase activity, nitric oxide synthase (NOS) activity, and cyclic guanosine monophosphate (cGMP) content were also assessed. Radial artery pressure pulse waveforms were used to track the hemodynamic actions of NTG. Three days of NTG/placebo was associated with a significant increase in platelet NO and O
2
·−
production from 1.0±1.17 to 2.52±0.88 pmol/10
8
platelets and 13.2±4.8 to 72.5±34.4 pmol/10
8
platelets, respectively (
P
<0.01 for both). These changes were accompanied by increased platelet NADH oxidase activity from 47.9±11.0 to 65.3±13.6 pmol O
2
·−
min/mg protein and cGMP content from 0.60±0.10 to 0.89±0.16 pmol/10
9
platelets (
P
<0.05 for both). Administration of NTG/ascorbate attenuated both NO and O
2
·−
release in platelets.

Conclusions

—

Three days of continuous transdermal administration of NTG was accompanied by increased platelet NO and O
2
·−
production and NADH oxidase activity that was suppressed by coadministration of oral ascorbate. Although a significant degree of tolerance would be expected during continuous nitrate administration, a residual hemodynamic action could be identified by arterial pulse contour analysis.
Integrated Analysis of Myocardial Blush and ST-Segment Elevation Recovery After Successful Primary Angioplasty
Authors: Poli Arnaldo , Fetiveau Raffaela , Vandoni Pietro , del Rosso Gianfranco , D’Urbano Maurizio , Seveso Giovanni , Cafiero Francesco , De Servi Stefano
Published: July 2002

Background

—

ST-segment elevation (ΣSTe) recovery and the angiographic myocardial blush (MB) grade are useful markers of microvascular reperfusion after recanalization of the infarct-related artery. We investigated the ability of a combined analysis of MB grade and ΣSTe changes to identify different patterns of myocardial reperfusion shortly after primary percutaneous coronary angioplasty (PTCA) and to predict 7-day and 6-month left ventricular (LV) functional recovery.

Methods and Results

—

MB grade and ΣSTe recovery were evaluated shortly after successful primary PTCA (restoration of TIMI grade 3 flow) in 114 consecutive patients with ΣSTe acute myocardial infarction. LV function was assessed by 2D echocardiograms before PTCA and at 7 days and 6 months thereafter. By combining MB and ΣSTe changes, 3 main groups of patients were identified. Group 1 patients (n=60) had both significant MB (grade 2 to 3) and ΣSTe recovery (>50% versus basal ΣSTe) and a high rate of 7-day (65%) and 6-month (95%) LV functional recovery. In group 2 patients (n=21), who showed MB but persistent ΣSTe, the prevalence of early LV functional recovery was low (24%) but increased up to 86% in the late phase. Group 3 patients (n=28), who had neither significant MB nor ΣSTe resolution, had poor early (18%) and late (32%) LV functional recovery.

Conclusions

—

After successful primary PTCA, integrated analysis of MB and ΣSTe recovery allows a real-time grading of microvascular reperfusion of the infarct area and predicts the time-course and magnitude of LV functional recovery.
Long-Term Endothelin A Receptor Blockade Inhibits Electrical Remodeling in Cardiomyopathic Hamsters
Authors: Matsumoto Yasunori , Aihara Hajime , Yamauchi-Kohno Rikako , Reien Yoshie , Ogura Takehiko , Yabana Hideo , Masuda Yoshiaki , Sato Toshiaki , Komuro Issei , Nakaya Haruaki
Published: July 2002

Background

—

The endothelin (ET) system is activated in failing hearts. Congestive heart failure frequently is associated with ventricular arrhythmias, which may result from electrical remodeling such as changes of ionic current density and heterogeneous action potential prolongation. We examined the effects of long-term ET
A
receptor blockade on the electrophysiological properties of ventricular cells, the surface ECG, and the survival in BIO 14.6 cardiomyopathic hamsters.

Methods and Results

—

Membrane currents and action potentials were recorded from left ventricular cells isolated from normal F1β hamsters and cardiomyopathic BIO 14.6 hamsters untreated and chronically treated with TA-0201, an ET
A
receptor antagonist. In ventricular cells of untreated BIO 14.6 hamsters, the action potential duration was prolonged and the densities of the L-type Ca
2+
current (
I
Ca,L
), the transient outward current (
I
to
), the delayed rectifier K
+
current (
I
K
), and the inward rectifier K
+
current (
I
K1
) were decreased compared with those of F1β hamsters. Long-term treatment with the ET
A
receptor antagonist significantly attenuated action potential duration prolongation and reduction of
I
to
,
I
K
, and
I
Ca,L
in BIO 14.6 ventricular cells. Long-term ET
A
receptor blockade prevented the QT prolongation and ventricular arrhythmias and improved the survival rate in the cardiomyopathic hamsters.

Conclusions

—

Long-term treatment with an ET
A
antagonist inhibits electrical remodeling such as downregulation of K
+
and Ca
2+
currents, action potential prolongation, and the increased QT interval and thereby suppresses ventricular arrhythmias in cardiomyopathic hearts. ET
A
receptor blockade may provide a new strategy for the prevention of ventricular arrhythmias associated with heart failure.
Mice Display Sex Differences in Halothane-Induced Polymorphic Ventricular Tachycardia
Authors: Drici Milou-Daniel , Baker Linda , Plan Patricia , Barhanin Jacques , Romey Georges , Salama Guy
Published: July 2002

Background

—

Molecularly engineered mice are extensively used as models of cardiovascular diseases, yet little is known about sex differences in the electrophysiology of mouse hearts.

Methods and Results

—

This study investigated the influence of sex on drug-induced polymorphic ventricular tachycardia (PVT) in Langendorff-perfused male and female mice hearts (n=54) by injecting a bolus of halothane (1.75 mmol/L) in the perfusate while recording ECGs or optical action potentials (APs). There were no statistically significant differences between male and female hearts (n=54) with respect to mean RR (193±5 ms), PR (47±1 ms), QT intervals (101±3 ms), optical AP durations (APD
75
=23.11±4.2 ms), dispersion of refractory periods, and conduction velocities (n=5 male and 5 female). Halothane induced PVTs lasting a mean duration of 90 seconds; in female hearts, 55% of PVTs lasted longer than the median, whereas in male hearts 17% exceeded the mean (
P
<0.05). The total duration of PVTs exposed a marked sex difference, 378±144 seconds in female versus 27±10 seconds in male hearts (
P
<0.05). In optically mapped male hearts, halothane reduced APD
75
(17.61±1.6 ms) and then elicited VTs (n=6 of 6), but in female hearts, halothane elicited PVTs (n=1 of 6) or arrested the hearts (n=5 of 6). Except for KCNE1, Northern blots (KCNQ1, MERG, Kv1.5, connexins 40 and 43, TREK1, and TASK1) did not detect sex differences.

Conclusions

—

This mouse model reveals sex difference in response to a pharmacological challenge yet does not display sex differences in standard electrophysiological parameters. Differences in KCNE1 may contribute to sex differences uncovered by halothane.
TAT Protein Transduction Into Isolated Perfused Hearts
Authors: Gustafsson Åsa B. , Sayen M. Richard , Williams Scott D. , Crow Michael T. , Gottlieb Roberta A.
Published: July 2002

Background

—

Linkage of the 11–amino-acid transduction domain of HIV TAT to a heterologous protein allows the protein to be transduced readily into cells.

Methods and Results

—

In this study, we inserted the apoptosis repressor with caspase recruitment domain (ARC) or β-galactosidase (β-gal) cDNA into the pTAT-hemagglutinin bacterial expression vector to produce genetic in-frame TAT-ARC or TAT-β-gal fusion proteins for use in cell culture and in Langendorff perfusion of adult rat hearts. TAT-β-gal and TAT-ARC were conjugated with Texas Red and could be detected in >95% of cells. TAT-ARC was able to protect H9c2 cells against cell death mediated by hydrogen peroxide, as measured by protection against the loss of mitochondrial membrane potential and preservation of nuclear morphology. Isolated adult hearts were perfused with recombinant TAT-β-gal or TAT-ARC (20 nmol/L) for 15 minutes and then subjected to 30 minutes of global no-flow ischemia, followed by 2 hours of reperfusion. Protein transduction was assessed by Western blotting of cell lysates and cytosolic and mitochondrial fractions and by fluorescence microscopy of Texas Red–conjugated TAT proteins. TAT-β-gal and TAT-ARC readily transduced into perfused hearts and were homogeneously distributed. Infarct size was determined by 2,3,5-triphenyltetrazolium chloride staining, and creatine kinase release was measured. Transduction of TAT-ARC was cardioprotective when administered before global ischemia and reperfusion.

Conclusions

—

Our results demonstrate that TAT-linked fusion protein transduction into the myocardium is feasible and that transduction of TAT-ARC is protective in cell culture and in the perfused heart.
Effects of Chronic Calcium Channel Blockade on Sympathetic Nerve Activity in Hypertension
Authors: Binggeli Christian , Corti Roberto , Sudano Isabella , Luscher Thomas F. , Noll Georg
Published: July 2002

The sympathetic nervous system (SNS) is an important regulator of the circulation. Its activity is increased in hypertension and heart failure and adversely affects prognosis. Although certain drugs inhibit SNS, dihydropyridine calcium antagonists may stimulate the system. Phenylalkylamine calcium antagonists such as verapamil have a different pharmacological profile. We therefore tested the hypothesis of whether amlodipine, nifedipine, or verapamil differs in the effects on muscle sympathetic nerve activity (MSA). Forty-three patients (31 men, 12 women) with mild to moderate hypertension were randomly assigned to 1 drug for 8 weeks. Blood pressure, heart rate, and MSA (by microneurography) were measured at baseline and after 8 weeks of treatment. All calcium antagonists led to a similar decrease in blood pressure of 5.0±1.5 to 6.4±1.4 mm Hg at 8 weeks (
P
<0.001 versus baseline). There were no significant differences in MSA between groups. With amlodipine, MSA averaged 49±3 bursts/min (3 versus baseline); with nifedipine, 48±3 bursts/min (2 versus baseline); and with verapamil, 49±2 bursts/min (all,
P
=NS). With verapamil, norepinephrine decreased by 4% but tended to increase by about one third with amlodipine or nifedipine (
P
=NS). Thus, in hypertension slow release forms of verapamil, nifedipine, and amlodipine exert comparable antihypertensive effects and do not change MSA, although there was a trend toward decreased MSA and plasma norepinephrine with verapamil.
Angiotensin II Type 2 Receptor Gene Transfer Downregulates Angiotensin II Type 1a Receptor in Vascular Smooth Muscle Cells
Authors: Jin Xue-Qing , Fukuda Noboru , Su Jin-Zi , Lai Yi-Mu , Suzuki Ryo , Tahira Yoshiko , Takagi Hiroto , Ikeda Yukihiro , Kanmatsuse Katsuo , Miyazaki Hitoshi
Published: July 2002

Two distinct subtypes of angiotensin (Ang) II receptors, type 1 (AT
1
) and type 2 (AT
2
), have been identified. Vascular smooth muscle cells (VSMCs) usually express AT
1
receptor. To elucidate the direct effects of the AT
2
receptor on the AT
1
receptor in VSMCs, we transfected AT
2
receptor gene into cultured rat VSMCs. Overexpression of AT
2
receptor significantly decreased expression of AT
1a
receptor at both the mRNA and protein levels in the presence and absence of Ang II in VSMCs. Overexpression of AT
2
receptor increased expression of bradykinin and inducible NO in the presence and absence of Ang II in VSMCs. Bradykinin B
2
receptor antagonist HOE–140 and NO synthase inhibitor

N
ω

-nitro-
l
-arginine methyl ester (L-NAME) inhibited the decreases in AT
1a
receptor expression by the overexpression of AT
2
receptor in VSMCs.
l
-Arginine augmented the decrease in AT
1a
receptor expression. Overexpression of AT
2
receptor suppressed basal DNA synthesis and proliferation of VSMCs and abolished response of DNA synthesis to Ang II in VSMCs. Our results demonstrate that overexpression of the AT
2
receptor downregulates AT
1a
receptor expression in rat VSMCs in a ligand-independent manner that is mediated by the bradykinin/NO pathway. Downregulation of AT
1a
receptor is a novel mechanism by which the AT
2
receptor regulates growth and metabolism of VSMCs.
Plasma ProANP
1–30
Reflects Salt Sensitivity in Subjects With Heredity for Hypertension
Authors: Melander Olle , Frandsen Erik , Groop Leif , Hulthén U. Lennart
Published: July 2002

The aim of the present study was to investigate whether plasma concentration of proANP
1–30
, the N-terminal fragment of the atrial natriuretic peptide prohormone, or 24-hour urinary excretion of urodilatin reflects the degree of salt sensitivity in hypertension-prone individuals. Plasma concentration of proANP
1–30
and urinary urodilatin excretion were determined at baseline, after 1 week on a low-salt diet (10 mmol/d) and after another week on a high-salt diet (240 mmol/d) in 30 healthy subjects with heredity for hypertension. Salt sensitivity was defined as the difference between mean arterial blood pressure after the high-salt diet and the mean arterial blood pressure after the low-salt diet. High- versus low-salt intake increased proANP
1–30
(668±330 versus 358±150 pmol/L;
P
<0.00001) and urodilatin (18.7±5.2 versus 16.0±8.3 pmol/24 h;
P
<0.05). ProANP
1–30
correlated with salt sensitivity at baseline (
r
=0.76,
P
<0.000001), after the low- (
r
=0.80,
P
<0.0000001) and high-salt diets (
r
=0.85,
P
<0.00000001). The increase in proANP
1–30
induced by changing from the low- to the high-salt diet was also directly related to salt sensitivity (
r
=0.78,
P
<0.000001). ProANP
1–30
was not related to urinary sodium excretion. Neither urodilatin nor the sodium-induced change in urodilatin correlated with salt sensitivity. However, urodilatin was related to the urinary sodium excretion at baseline (
r
=0.58,
P
<0.01) and after the high-salt diet (
r
=0.62,
P
<0.001). In conclusion, the close correlations between proANP
1–30
and salt sensitivity suggest that proANP
1–30
may serve as a marker for salt sensitivity and could be useful in identifying subjects who would benefit from dietary salt restriction to prevent development of hypertension.
Differential Regulation of Elevated Renal Angiotensin II in Chronic Renal Ischemia
Authors: Tokuyama Hirobumi , Hayashi Koichi , Matsuda Hiroto , Kubota Eiji , Honda Masanori , Okubo Ken , Takamatsu Ichiro , Tatematsu Satoru , Ozawa Yuri , Wakino Shu , Saruta Takao
Published: July 2002

The present study was undertaken to clarify the role of intrarenal angiotensin (Ang) II and its generating pathways in clipped and nonclipped kidneys of 4-week unilateral renal artery stenosis in anesthetized dogs. After 4 weeks, renal plasma flow (RPF) decreased in clipped and nonclipped kidneys (baseline, 59±3; clipped, 16±1; nonclipped, 44±2 mL/min;
P
<0.01, n=22). Renal Ang I levels increased only in clipped, whereas intrarenal Ang II contents were elevated in both clipped (from 0.7±0.1 to 2.0±0.2 pg/mg tissue) and nonclipped kidneys (from 0.6±0.1 to 2.5±0.3 pg/mg tissue). Intrarenal ACE activity was increased in nonclipped kidneys but was unaltered in clipped kidneys. An angiotensin receptor antagonist (olmesartan medoxomil) given into the renal artery markedly restored RPF, and dilated both afferent and efferent arterioles (using intravital videomicroscopy). Furthermore, in clipped kidneys, the elevated Ang II was suppressed by a chymase inhibitor, chymostatin (from 2.1±0.6 to 0.8±0.1 pg/mg tissue;
P
<0.05), but not by cilazaprilat. In nonclipped kidneys, in contrast, cilazaprilat, but not chymostatin, potently inhibited the intrarenal Ang II generation (from 2.4±0.3 to 1.5±0.2 pg/mg tissue;
P
<0.05). Finally, [Pro
11
-
d
-Ala
12
]Ang I (an inactive precursor that yields Ang II by chymase but not by ACE; 1 to 50 nmol/kg) markedly elevated intrarenal Ang II in clipped, but not in nonclipped, kidneys. In conclusion, renal Ang II contents were elevated in both clipped and nonclipped kidneys, which contributed to the altered renal hemodynamics and microvascular tone. Furthermore, the mechanisms for intrarenal Ang II generation differ, and chymase activity is enhanced in clipped kidneys, whereas ACE-mediated Ang II generation is possibly responsible for elevated Ang II contents in nonclipped kidneys.
Brain-Selective Overexpression of Angiotensin (AT
1
) Receptors Causes Enhanced Cardiovascular Sensitivity in Transgenic Mice
Authors: Lazartigues Eric , Dunlay Shannon M. , Loihl Angela K. , Sinnayah Puspha , Lang Julie A. , Espelund Joshua J. , Sigmund Curt D. , Davisson Robin L.
Published: July 2002

To examine the physiological importance of brain angiotensin II type 1 (AT
1
) receptors, we developed a novel transgenic mouse model with rat AT
1a
receptors targeted selectively to neurons of the central nervous system (CNS). A transgene consisting of 2.8 kb of the rat neuron-specific enolase (NSE) 5′ flanking region fused to a cDNA encoding the full open-reading frame of the rat AT
1a
receptor was constructed and transgenic mice (NSE-AT
1a
) were generated. Two of six transgenic founder lines exhibited brain-selective expression of the transgene at either moderate or high levels. Immunohistochemistry revealed widespread distribution of AT
1
receptors in neurons throughout the CNS. This neuron-targeted overexpression of AT
1a
receptors resulted in enhanced cardiovascular responsiveness to intracerebroventricular (ICV) angiotensin II (Ang II) injection but not to other central pressor agents, demonstrating functional overexpression of the transgene in NSE-AT
1a
mice. Interestingly, baseline blood pressure (BP) was not elevated in either transgenic line. However, blockade of central AT
1
receptors with ICV losartan caused significant falls in basal BP in NSE-AT
1a
mice but had no effect in nontransgenic controls. These results suggest that whereas there is an enhanced contribution of central AT
1
receptors to the maintenance of baseline BP in NSE-AT
1a
mice, particularly effective baroreflex buffering prevents hypertension in this model. Used both independently, and in conjunction with mice harboring gene-targeted deletions of AT
1a
receptors, this new model will permit quantitative and relevant investigations of the role of central AT
1a
receptors in cardiovascular homeostasis in health and disease.
Inhibition of Protein Kinase Cα Prevents Endothelial Cell Migration and Vascular Tube Formation In Vitro and Myocardial Neovascularization In Vivo
Authors: Wang Aihong , Nomura Motohiro , Patan Sybill , Ware J. Anthony
Published: July 2002

Although protein kinase C (PKC) activation is required for endothelial cell (EC) growth, migration, adhesion, and vessel formation, the role of individual PKC isoenzymes in these events is not defined. Because PKCα has been previously linked with enhanced EC migration and response to angiogenic growth factors, we characterized a specific phosphorothioate-modified 21-mer antisense PKCα (AS-PKCα). AS-PKCα (500 nmol/L) prevented the expression of PKCα protein by 90% in human ECs and did not reduce the expression of any other PKC isoenzyme. AS-PKCα reduced human EC migration by 64% compared with its control oligonucleotide in a “scratch” wounding assay, and AS-PKCα reduced human EC adhesion to the extracellular matrix protein vitronectin by 18%. Phosphorylation of mitogen-activated protein kinase (extracellular signal–regulated kinase 1/2) induced by vascular endothelial growth factor was inhibited by 30% in human ECs transfected with AS-PKCα. Compared with control, AS-PKCα also reduced the number of EC tubes formed in a 3D type I collagen gel assay by 37.5%. Finally, using an osmotic minipump, we infused AS-PKCα into mice in which myocardial infarction was induced by coronary ligation and found that the oligonucleotide was primarily taken up by intramyocardial blood vessels. Compared with the results with control oligonucleotide, AS-PKCα oligonucleotide inhibited the number of anti-PKCα–stained blood vessels by 48% and reduced the total vessel number by 72% as well. In conclusion, the expression of PKCα is required for full EC migration, adhesion to vitronectin, vascular endothelial growth factor–induced extracellular signal–regulated kinase activation, and tube formation and is likely to be of importance in myocardial angiogenesis in vivo after ischemia.
Differential Association and Localization of Myosin Phosphatase Subunits During Agonist-Induced Signal Transduction in Smooth Muscle
Authors: Shin Heung-Mook , Je Hyun-Dong , Gallant Cynthia , Tao Terence C. , Hartshorne David J. , Ito Masaaki , Morgan Kathleen G.
Published: July 2002

It has been known for some time that agonist-induced contractions of vascular smooth muscle are often associated with a sensitization of the contractile apparatus to intracellular Ca
2+
. One mechanism that has been suggested to explain Ca
2+
sensitization is inhibition of myosin phosphatase activity. In the present study, we tested the hypothesis that differential localization of the phosphatase might be associated with its inhibition. Quantitative confocal microscopy of freshly dissociated, fully contractile smooth muscle cells was used in parallel with measurements of myosin light chain and myosin phosphatase phosphorylation. The results indicate that, in the smooth muscle cells, the catalytic and targeting subunits of the phosphatase are dissociated from each other in an agonist-specific manner and that the dissociation is accompanied by a slower rate of myosin phosphorylation. Targeting of myosin phosphatase to the cell membrane precedes the dissociation of subunits and is associated with phosphorylation of the targeting subunit at a Rho-associated kinase (ROK) phosphorylation site. The phosphorylation and membrane translocation of the targeting subunit are inhibited by a ROK inhibitor. This dissociation of subunits may provide a mechanism for the decreased phosphatase activity of phosphorylated myosin phosphatase.
Prourokinase Mutant That Induces Highly Effective Clot Lysis Without Interfering With Hemostasis
Authors: Liu Jian-Ning , Liu Jian-Xia , Liu Bei-fang , Sun Ziyong , Zuo Jian-Ling , Zhang Pei-xiang , Zhang Jing , Chen Yu-hong , Gurewich Victor
Published: July 2002

Prourokinase (proUK) is a zymogenic plasminogen activator that at pharmacological doses is prone to nonspecific activation to urokinase. This has handicapped therapeutic exploitation of its fibrin-specific physiological properties. To attenuate this susceptibility without compromising specific activation of proUK on a fibrin clot, a Lys300→His mutation (M5) was developed. M5 had a lower intrinsic activity and, therefore, remained stable in plasma at a 4-fold higher concentration than did proUK. M5 had a higher 2-chain activity and induced more rapid plasminogen activation and fibrin-specific clot lysis in vitro. Sixteen dogs embolized with radiolabeled clots were infused with saline, proUK, tissue plasminogen activator, or M5. The lower intrinsic activity allowed a higher infusion rate with M5, which induced the most rapid and efficient clot lysis (50% clot lysis by ≈600 μg/kg M5 versus ≈1200 μg/kg proUK). In association with this, M5 caused neither a significant increase in the primary bleeding time nor secondary bleeding (total blood loss). By contrast, these measurements increased 4-fold and 5-fold, respectively, with proUK and >5-fold and 8-fold, respectively, with tissue plasminogen activator. Clot lysis by M5 and hemostasis were further evaluated in 6 rhesus monkeys. M5 again induced rapid clot lysis without a significant increase in the primary bleeding time, and secondary bleeding did not occur. In conclusion, a site-directed mutation designed to improve the stability of proUK in blood at therapeutic concentrations induced superior clot lysis in vitro and in vivo without causing significant interference with hemostasis.
Inhibition of Na
+
-H
+
Exchange Prevents Hypertrophy, Fibrosis, and Heart Failure in β
1
-Adrenergic Receptor Transgenic Mice
Authors: Engelhardt Stefan , Hein Lutz , Keller Ursula , Klämbt Kerstin , Lohse Martin J.
Published: July 2002

Chronic stimulation of the β
1
-adrenergic receptor leads to hypertrophy and heart failure in β
1
-adrenergic receptor transgenic mice and contributes to disease progression in heart failure patients. The cellular mechanisms underlying these detrimental effects are largely unknown. In this study, we have identified the cardiac Na
+
-H
+
exchanger (NHE1) as a novel mediator of adrenergically induced heart failure. β
1
-Adrenergic receptor transgenic mice showed upregulation of both NHE1 mRNA (+140±6%) and protein (+42±19%). In order to test whether increased NHE1 is causally related to β
1
-adrenergic–induced hypertrophy, fibrosis, and heart failure, β
1
-adrenergic receptor transgenic (TG) and wild-type (WT) littermates were treated with a diet containing 6000 ppm of the NHE1 inhibitor cariporide or control chow for 8 months. There was significant hypertrophy of cardiac myocytes in β
1
-adrenergic receptor transgenic mice (2.3-fold increase in myocyte cross-sectional area), which was virtually absent in cariporide-fed animals. Interstitial fibrosis was prominent throughout the left ventricular wall in nontreated β
1
-adrenergic receptor transgenic mice (4.8-fold increase in collagen volume fraction); cariporide treatment completely prevented this development of fibrosis. Left ventricular catheterization showed that cariporide also prevented the loss of contractile function in β
1
-adrenergic receptor transgenic mice: whereas untreated transgenic mice showed a significant decrease in left ventricular contractility (5250±570 mm Hg/s TG versus 7360±540 mm Hg/s WT, dp/dt
max
), this decrease was completely prevented by cariporide (8150±520 mm Hg/s TG cariporide). Inhibition of NHE1 prevented the development of heart failure in β
1
-receptor transgenic mice. We conclude that the cardiac Na
+
-H
+
exchanger 1 is essential for the detrimental cardiac effects of chronic β
1
-receptor stimulation in the heart.
Influence of Mitochondrial Inhibition on Global and Local [Ca
2+
]
i
in Rat Tail Artery
Authors: Swärd Karl , Dreja Karl , Lindqvist Anders , Persson Erik , Hellstrand Per
Published: July 2002

Inhibition of oxidative metabolism is often found to decrease contractility of systemic vascular smooth muscle, but not to reduce global [Ca
2+
]
i
. In the present study, we probe the hypothesis that it is associated with an altered pattern of intracellular Ca
2+
oscillations (waves) influencing force development. In the rat tail artery, mitochondrial inhibitors (rotenone, antimycin A, and cyanide) reduced α
1
-adrenoceptor–stimulated force by 50% to 80%, but did not reduce global [Ca
2+
]
i
. Less relaxation (about 30%) was observed after inhibition of myosin phosphatase activity with calyculin A, suggesting that part of the metabolic sensitivity involves the regulation of myosin 20-kDa light chain phosphorylation, although no decrease in phosphorylation was found in freeze-clamped tissue. Confocal imaging revealed that the mitochondrial inhibitors increased the frequency but reduced the amplitude of asynchronous cellular Ca
2+
waves elicited by α
1
stimulation. The altered wave pattern, in association with increased basal [Ca
2+
]
i
, accounted for the unchanged global [Ca
2+
]
i
. Inhibition of glycolytic ATP production by arsenate caused similar effects on Ca
2+
waves and global [Ca
2+
]
i
, developing gradually in parallel with decreased contractility. Inhibition of wave activity by the InsP
3
receptor antagonist 2-APB correlated closely with relaxation. Furthermore, abolition of waves with thapsigargin in the presence of verapamil reduced force by about 50%, despite unaltered global [Ca
2+
]
i
, suggesting that contraction may at least partly depend on Ca
2+
wave activity. This study therefore indicates that mitochondrial inhibition influences Ca
2+
wave activity, possibly due to a close spatial relationship of mitochondria and the sarcoplasmic reticulum and that this contributes to metabolic vascular relaxation.
Dual Actions of the Gα
q
Agonist
Pasteurella multocida
Toxin to Promote Cardiomyocyte Hypertrophy and Enhance Apoptosis Susceptibility
Authors: Sabri Abdelkarim , Wilson Brenda A. , Steinberg Susan F.
Published: July 2002

Previous attempts to delineate the consequences of Gα
q
activation in cardiomyocytes relied largely on molecular strategies in cultures or transgenic mice. Modest levels of wild-type Gα
q
overexpression induce stable cardiac hypertrophy, whereas intense Gα
q
stimulation induces cardiomyocyte apoptosis. The precise mechanism(s) whereby traditional targets of Gα
q
subunits that induce hypertrophy also trigger cardiomyocyte apoptosis is not obvious and is explored with recombinant
Pasteurella multocida
toxin (rPMT, a Gα
q
agonist). Cells cultured with rPMT display cardiomyocyte enlargement, sarcomeric organization, and increased atrial natriuretic factor expression in association with activation of phospholipase C, novel protein kinase C (PKC) isoforms, extracellular signal-regulated protein kinase (ERK), and (to a lesser extent) JNK/p38-MAPK. rPMT stimulates the ERK cascade via epidermal growth factor (EGF) receptor transactivation in cardiac fibroblasts, but EGF receptor transactivation plays no role in ERK activation in cardiomyocytes. Surprisingly, rPMT (or novel PKC isoform activation by PMA) decreases basal Akt phosphorylation; rPMT prevents Akt phosphorylation by EGF or IGF-1 and functionally augments cardiomyocyte apoptosis in response to H
2
O
2
. These results identify a Gα
q
-PKC pathway that represses basal Akt phosphorylation and impairs Akt stimulation by survival factors. Because inhibition of Akt enhances cardiomyocyte susceptibility to apoptosis, this pathway is predicted to contribute to the transition from hypertrophy to cardiac decompensation and could be targeted for therapy in heart failure.
Angiotensin II Induces Myocyte Enhancer Factor 2- and Calcineurin/Nuclear Factor of Activated T Cell-Dependent Transcriptional Activation in Vascular Myocytes
Authors: Suzuki Etsu , Nishimatsu Hiroaki , Satonaka Hiroshi , Walsh Kenneth , Goto Atsuo , Omata Masao , Fujita Toshiro , Nagai Ryozo , Hirata Yasunobu
Published: July 2002

It is well known that angiotensin II (Ang II) is implicated in the phenotypic modulation and hypertrophy of vascular smooth muscle cells (VSMCs). To study the mechanisms by which Ang II contributes to the pathological changes of VSMCs, we examined whether Ang II stimulated myocyte enhancer factor 2 (MEF2)- and calcineurin/nuclear factor of activated T cell (NFAT)-dependent transcriptional activation of genes in VSMCs. Ang II increased the DNA binding activity of MEF2A and its expression at the protein level. Ang II induced c-
jun
promoter activity, and this increase was inhibited by dominant-negative mutants of MEF2A and mitogen-activated protein kinase kinase 6 but not by calcineurin inhibitors. Ang II stimulated NFAT DNA binding activity and NFAT-dependent gene transcription, and these effects of Ang II were inhibited by calcineurin inhibitors. Furthermore, Ang II induced the promoter activity of the nonmuscle-type myosin heavy chain B gene, which we used as a marker of the dedifferentiated state of VSMCs, and this increase was inhibited by calcineurin inhibitors but not by the dominant-negative mutants of MEF2A or mitogen-activated protein kinase kinase 6. Finally, Ang II increased protein synthesis, and this increase was inhibited by infection with an adenovirus construct that expresses the dominant-negative mutant of MEF2A but not by calcineurin inhibitors. These results suggest that Ang II stimulates the MEF2- and calcineurin/NFAT-dependent pathways and that these pathways have distinct roles in VSMCs.
PKCζ Regulates TNF-α–Induced Activation of NADPH Oxidase in Endothelial Cells
Authors: Frey Randall S. , Rahman Arshad , Kefer John C. , Minshall Richard D. , Malik Asrar B.
Published: July 2002

Although oxidant generation by NADPH oxidase is known to play an important role in signaling in endothelial cells, the basis of activation of NADPH oxidase is incompletely understood. The atypical isoform of protein kinase C, PKCζ, has been implicated in the mechanism of tumor necrosis factor-α (TNF-α)–induced oxidant generation in endothelial cells; thus, in the present study, we have addressed the role of PKCζ in regulating NADPH oxidase function. We showed by immunoblotting and confocal microscopy the presence of the major cytosolic NADPH oxidase subunits, p47

phox

and membrane-bound gp91

phox

in human pulmonary artery endothelial (HPAE) cells. TNF-α failed to activate oxidant generation in lung vascular endothelial cells derived from
p47

phox
−/−

and
gp91

phox
−/−

mice, indicating the requirement of NADPH oxidase in mediating the oxidant generation in endothelial cells. Stimulation of HPAE cells with TNF-α resulted in the phosphorylation of p47

phox

and its association with gp91

phox

. Inhibition of PKCζ by multiple pharmacological and genetic approaches prevented the TNF-α–induced phosphorylation of p47

phox

, and its translocation to the membrane. PKCζ was shown to colocalize with p47

phox

, and inhibition of PKCζ activation prevented the interaction of p47

phox

with gp91

phox

induced by TNF-α. Furthermore, inhibition of association of p47

phox

with gp91

phox

prevented the oxidant generation in endothelial cells. These data demonstrate a novel function of PKCζ in signaling oxidant generation in endothelial cells by the activation of NADPH oxidase, which may be important in mediating endothelial activation responses.
14,15-Epoxyeicosa-5(
Z
)-enoic Acid
Authors: Gauthier Kathryn M. , Deeter Christina , Krishna U. Murali , Reddy Y. Krishna , Bondlela Muralidhar , Falck J.R. , Campbell William B.
Published: July 2002

Endothelium-dependent hyperpolarization and relaxation of vascular smooth muscle are mediated by endothelium-derived hyperpolarizing factors (EDHFs). EDHF candidates include cytochrome P-450 metabolites of arachidonic acid, K
+
, hydrogen peroxide, or electrical coupling through gap junctions. In bovine coronary arteries, epoxyeicosatrienoic acids (EETs) appear to function as EDHFs. A 14,15-EET analogue, 14,15-epoxyeicosa-5(
Z
)-enoic acid (14,15-EEZE) was synthesized and identified as an EET-specific antagonist. In bovine coronary arterial rings preconstricted with U46619, 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET induced concentration-related relaxations. Preincubation of the arterial rings with 14,15-EEZE (10 μmol/L) inhibited the relaxations to 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET but was most effective in inhibiting 14,15-EET–induced relaxations. 14,15-EEZE also inhibited indomethacin-resistant relaxations to methacholine and arachidonic acid and indomethacin-resistant and
l
-nitroarginine-resistant relaxations to bradykinin. It did not alter relaxation responses to sodium nitroprusside, iloprost, or the K
+
channel activators (NS1619 and bimakalim). Additionally, in small bovine coronary arteries pretreated with indomethacin and
l
-nitroarginine and preconstricted with U46619, 14,15-EEZE (3 μmol/L) inhibited bradykinin (10 nmol/L)–induced smooth muscle hyperpolarizations and relaxations. In rat renal microsomes, 14,15-EEZE (10 μmol/L) did not decrease EET synthesis and did not alter 20-hydroxyeicosatetraenoic acid synthesis. This analogue acts as an EET antagonist by inhibiting the following: (1) EET-induced relaxations, (2) the EDHF component of methacholine-induced, bradykinin-induced, and arachidonic acid–induced relaxations, and (3) the smooth muscle hyperpolarization response to bradykinin. Thus, a distinct molecular structure is required for EET activity, and alteration of this structure modifies agonist and antagonist activity. These findings support a role of EETs as EDHFs.
Impairment of Store-Operated Ca
2+
Entry in TRPC4
−/−
Mice Interferes With Increase in Lung Microvascular Permeability
Authors: Tiruppathi Chinnaswamy , Freichel Marc , Vogel Stephen M. , Paria Biman C. , Mehta Dolly , Flockerzi Veit , Malik Asrar B.
Published: July 2002

We investigated the possibility that the TRPC gene family of putative store-operated Ca
2+
entry channels contributes to the increase in microvascular endothelial permeability by prolonging the rise in intracellular Ca
2+
signaling. Studies were made in wild-type (wt) and TRPC4 knockout (TRPC4
−/−
) mice and lung vascular endothelial cells (LECs) isolated from these animals. RT-PCR showed expression of TRPC1, TRPC3, TRPC4, and TRPC6 mRNA in wt LECs, but TRPC4 mRNA expression was not detected in TRPC4
−/−
LECs. We studied the response to thrombin because it is known to increase endothelial permeability by the activation of G protein-coupled proteinase-activated receptor-1 (PAR-1). In wt LECs, thrombin or PAR-1 agonist peptide (TFLLRNPNDK-NH
2
) resulted in a prolonged Ca
2+
transient secondary to influx of Ca
2+
. Ca
2+
influx activated by thrombin was blocked by La
3+
(1 μmol/L). In TRPC4
−/−
LECs, thrombin or TFLLRNPNDK-NH
2
produced a similar initial increase of intracellular Ca
2+
secondary to Ca
2+
store depletion, but Ca
2+
influx induced by these agonists was drastically reduced. The defect in Ca
2+
influx in TRPC4
−/−
endothelial cells was associated with lack of thrombin-induced actin-stress fiber formation and a reduced endothelial cell retraction response. In isolated-perfused mouse lungs, the PAR-1 agonist peptide increased microvessel filtration coefficient (K
f,c
), a measure of vascular permeability, by a factor of 2.8 in wt and 1.4 in TRPC4
−/−
; La
3+
(1 μmol/L) addition to wt lung perfusate reduced the agonist effect to that observed in TRPC4
−/−
. These results show that TRPC4-dependent Ca
2+
entry in mouse LECs is a key determinant of increased microvascular permeability.