bluuebunny/crossref_metadata_2025 huggingface dataset:
Prx1 Controls Vascular Smooth Muscle Cell Proliferation and Tenascin-C Expression and Is Upregulated With Prx2 in Pulmonary Vascular Disease
Authors: Jones Frederick S. , Meech Robyn , Edelman David B. , Oakey Rebecca J. , Jones Peter Lloyd
Published: September 2007

Prx1 and Prx2 are homeobox transcription factors expressed during vasculogenesis. To begin to elucidate how Prx1 and Prx2 are regulated and function in the adult vasculature, in situ hybridization studies were performed. Prx1 and Prx2 mRNAs were not detected in normal adult rat pulmonary arteries; however, both genes were induced with vascular disease, colocalizing to sites of tenascin-C (TN-C) expression. Because catabolism of the extracellular matrix (ECM) is a critical step in the development of vascular disease, we investigated whether changes in vascular smooth muscle cell (SMC)–ECM interactions regulate Prx1 and Prx2. A10 SMCs cultured on native type I collagen showed low levels of Prx1 and Prx2 mRNA expression, whereas cells cultured on denatured collagen showed higher levels of expression of both genes. At a functional level, transfection of SMCs with a Prx1 expression plasmid significantly increased their growth. Because TN-C also promotes SMC growth and its expression is also upregulated by denatured collagen, we tested and thereafter showed that Prx1 expression significantly enhances TN-C gene promoter activity 20-fold. Similar experiments conducted with truncated Prx1 proteins showed that the N-terminal portion and the homeodomain of Prx1 were necessary to induce the bulk of TN-C promoter activity. These findings support the hypothesis that Prx genes are regulated by changes in SMC adhesion and play key morphoregulatory roles during the development and progression of pulmonary vascular disease in adults.
Homozygous
SCN5A
Mutation in Long-QT Syndrome With Functional Two-to-One Atrioventricular Block
Authors: Lupoglazoff J.M. , Cheav T. , Baroudi G. , Berthet M. , Denjoy I. , Cauchemez B. , Extramiana F. , Chahine M. , Guicheney P.
Published: September 2007

Abstract—

Heterozygous mutations in genes encoding cardiac ionic channel subunits
KCNQ1
,
HERG
,
SCN5A
,
KCNE1
, and
KCNE2
are causally involved in the dominant form of long-QT syndrome (LQTS) while homozygous mutations in
KCNQ1
and
KCNE1
cause LQTS with or without congenital deafness. In addition, two homozygous
HERG
mutations have been associated with severe LQTS with functional atrioventricular conduction anomalies in young children. A 2:1 atrioventricular block (AVB) with a major QTc prolongation (526 ms) was evidenced in a 5-year-old boy referred for syncope and seizure. LQTS was diagnosed and beta-blocking therapy initiated leading to normal atrioventricular conduction. Electrophysiological study provided support that location of the AVB was infra-Hisian. DNA analysis was performed in the proband and in asymptomatic family members. A novel missense mutation, V1777M, in the early C-terminal domain of
SCN5A
was identified. The proband was homozygous while the parents and two siblings were heterozygous carriers. Homozygote and heterozygote expression of the mutant channels in tsA201 mammalian cells resulted in a persistent inward sodium current of 3.96±0.83% and 1.49±0.47% at −30 mV, respectively, which was dramatically reduced in the presence of tetrodotoxin. This study provides the first evidence for a homozygous missense mutation in
SCN5A
and suggests that LQTS with functional 2:1 AVB in young children, a severe phenotype associated with bad prognosis, may be caused by homozygous or heterozygous compound mutations not only in
HERG
but also in
SCN5A
. The full text of this article is available at http://www.circresaha.org.
Mechanisms of Telomerase Induction During Vascular Smooth Muscle Cell Proliferation
Authors: Minamino T. , Kourembanas S.
Published: September 2007

Telomeres are primarily controlled by a highly specialized DNA polymerase termed telomerase. Recent studies have demonstrated that introduction of the telomerase catalytic component (TERT) into telomerase-negative cells activates telomerase and extends cell life span, whereas mice lacking telomerase activity revealed impaired cell proliferation in some organs as well as reduced tumorigenesis. These reports suggest that telomerase plays an important role in long-term cell viability and cell proliferation. However, the mechanism or mechanisms by which telomerase is induced or regulated remains to be elucidated. We report here that primary vascular smooth muscle cells (VSMCs) express telomerase and that increased telomerase activity correlates with cell proliferation. Inhibition of telomerase diminished growth of VSMCs, which suggests a crucial role for telomerase activation in the regulation of VSMC proliferation. We propose a novel model whereby telomerase is first activated in the cytoplasm before cell proliferation, followed by accumulation of activity in the nucleus during the logarithmic phase of cell growth. Activation of telomerase in VSMCs was linked to phosphorylation of TERT. The protein kinase inhibitor H7 suppressed the activation of telomerase in the cytoplasm and also inhibited the accumulation of TERT as well as telomerase activity in the nucleus. These data suggest that posttranslational modification of TERT by phosphorylation is important for activation and accumulation of telomerase into the nucleus in the process of VSMC proliferation.
Acid Prohormone Sequence Determines Size, Shape, and Docking of Secretory Vesicles in Atrial Myocytes
Authors: Baertschi Alex J. , Monnier Dominique , Schmidt Uta , Levitan Edwin S. , Fakan Stanislas , Roatti Angela
Published: September 2007

How vesicles are born in the
trans
-Golgi network and reach their docking sites at the plasma membrane is still largely unknown and is investigated in the present study on live, primary cultured atrial cardiomyocytes. Secretory vesicles (n=422) are visualized by expressing fusion proteins of proatrial natriuretic peptide (proANP) and green fluorescent protein. Myocytes expressing fusion proteins with intact proANP display two populations of fluorescent vesicles with apparent diameters of 120 and 175 nm, moving at a top velocity of 0.3 μm/s. The number of docked vesicles is significantly correlated with the number of mobile vesicles (
r
=0.71,
P
<0.0005). The deletion of the acidic N-terminal proANP[1-44] or point mutations (glu
23,24
→gln
23,24
) change size and shape—but not velocity—of the vesicles, and, strikingly, abolish their docking at the plasma membrane. The shapes thus change from spheres to larger, irregular floppy bags or vesicle trains. Deletion of the C-terminal proANP[45-127], where the ANP and its disulfide bond reside, does not change size, shape, docking, or velocity of the mobile vesicles. The N-terminal acid calcium-binding sequence of proANP is known to cause protein aggregation at the high calcium concentration prevailing in the
trans
-Golgi network. Therefore, these results indicate that amino acid residues favoring cargo aggregation are critically important in shaping the secretory vesicles and determining their fate—docking or not docking—at the plasma membrane. The full text of this article is available at http://www.circresaha.org.
A TREK-1–Like Potassium Channel in Atrial Cells Inhibited by β-Adrenergic Stimulation and Activated by Volatile Anesthetics
Authors: Terrenoire Cécile , Lauritzen Inger , Lesage Florian , Romey Georges , Lazdunski Michel
Published: September 2007

Many members of the two-pore-domain potassium (K
+
) channel family have been detected in the mammalian heart but the endogenous correlates of these channels still have to be identified. We investigated whether
I
KAA
, a background K
+
current activated by negative pressure (stretch) and by arachidonic acid (AA) and sensitive to intracellular acidification, could be the native correlate of TREK-1 in adult rat atrial cells. Using the inside-out configuration of the patch-clamp technique, we found that
I
KAA
, like TREK-1, was outwardly rectifying in physiological K
+
conditions, with a conductance of 41 pS at +50 mV. Like TREK-1,
I
KAA
was reversibly activated by clinical concentrations of volatile anesthetics (in mmol/L, chloroform 0.18, halothane 0.11, and isoflurane 0.69). In cell-attached experiments,
I
KAA
was inhibited by chlorophenylthio-cAMP (500 μmol/L) and also by stimulation of β-adrenergic receptors with isoproterenol (1 μmol/L). In addition, TREK-1 mRNAs were detected in all cardiac tissues, and the TREK-1 protein was immunolocalized in isolated atrial myocytes. Such a background potassium channel might contribute to the positive inotropic effects produced by β-adrenergic stimulation of the heart. It might also be involved in the regulation of the atrial natriuretic peptide secretion.
Role for Endothelin-1–Induced Superoxide and Peroxynitrite Production in Rebound Pulmonary Hypertension Associated With Inhaled Nitric Oxide Therapy
Authors: Wedgwood Stephen , McMullan D. Michael , Bekker Janine M. , Fineman Jeffrey R. , Black Stephen M.
Published: September 2007

Our previous studies have demonstrated that inhaled nitric oxide (NO) decreases nitric oxide synthase (NOS) activity in vivo and that this inhibition is associated with rebound pulmonary hypertension upon acute withdrawal of inhaled NO. We have also demonstrated that inhaled NO elevates plasma endothelin-1 (ET-1) levels and that pretreatment with PD156707, an ETA receptor antagonist, blocks the rebound hypertension. The objectives of this study were to further elucidate the role of ET-1 in the rebound pulmonary hypertension upon acute withdrawal of inhaled NO. Inhaled NO (40 ppm) delivered to thirteen 4-week-old lambs decreased NOS activity by 36.2% in control lambs (
P
<0.05), whereas NOS activity was preserved in PD156707-treated lambs. When primary cultures of pulmonary artery smooth muscle cells were exposed to ET-1, superoxide production increased by 33% (
P
<0.05). This increase was blocked by a preincubation with PD156707. Furthermore, cotreatment of cells with ET-1 and NO increased peroxynitrite levels by 26% (
P
<0.05), whereas preincubation of purified human endothelial nitric oxide synthase (eNOS) protein with peroxynitrite generated a nitrated enzyme with 50% activity relative to control (
P
<0.05). Western blot analysis of peripheral lung extracts obtained after 24 hours of inhaled NO revealed a 90% reduction in 3-nitrotyrosine residues (
P
<0.05) in PD156707-treated lambs. The nitration of eNOS was also reduced by 40% in PD156707-treated lambs (
P
<0.05). These data suggest that the reduction of NOS activity associated with inhaled NO therapy may involve ETA receptor–mediated superoxide production. ETA receptor antagonists may prevent rebound pulmonary hypertension by protecting endogenous eNOS activity during inhaled NO therapy.
Novel Competitive Inhibitor of NAD(P)H Oxidase Assembly Attenuates Vascular O2−and Systolic Blood Pressure in Mice
Authors: Rey F. E. , Cifuentes M. E. , Kiarash A. , Quinn M. T. , Pagano P. J.
Published: September 2007

We previously reported enhanced expression of the p67phoxand gp91phoxcomponents of NAD(P)H oxidase in angiotensin (Ang) II–induced hypertension, suggesting de novo assembly in response to Ang II. To examine the direct involvement of NAD(P)H oxidases in Ang II–induced O2−production, we designed a chimeric peptide that inhibits p47phoxassociation with gp91phoxin NAD(P)H oxidase (gp91ds-tat). This was achieved by linking a 9-amino acid peptide (aa) derived from HIV-coat protein (tat) to a 9-aa sequence of gp91phox(known to interact with p47phox). As a control, we constructed a chimera containingtatand a scrambled gp91 sequence (scramb-tat). We found that gp91ds-tatdecreased O2−levels in aortic rings treated with Ang II (10 pmol/L) but had no effect on either the O2−-generating enzyme xanthine oxidase or potassium superoxide–generated O2−. We infused vehicle, Ang II (0.75 mg · kg−1· d−1), Ang II+gp91ds-tat(10 mg · kg−1· d−1), or Ang II+scramb-tatintraperitoneally in C57Bl/6 mice and measured systolic blood pressure (SBP) on days 0, 3, 5, and 7 of infusion. SBP increased by day 3 in mice given Ang II and Ang II+scramb-tatbut was significantly lower with Ang II+gp91-tat. On day 7, SBP was still significantly inhibited in mice given Ang II+gp91ds-tat, whereas Ang II–induced O2−production was inhibited throughout the aorta as detected by dihydroethidium staining, consistent with the ability of this inhibitor to block the various vascular NAD(P)H oxidase isoforms. These data support the hypothesis that inhibition of the interaction of p47phoxand gp91phox(or its homologues) can block O2−production and attenuate blood pressure elevation in mice.
Angiopoietin-1 Reduces VEGF-Stimulated Leukocyte Adhesion to Endothelial Cells by Reducing ICAM-1, VCAM-1, and E-Selectin Expression
Authors: Kim Injune , Moon Sang-Ok , Park Sung Kwang , Chae Soo Wan , Koh Gou Young
Published: September 2007

Vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1) are potent vasculogenic and angiogenic factors that hold promise as a means to produce therapeutic vascularization and angiogenesis. However, VEGF also acts as a proinflammatory cytokine by inducing adhesion molecules that bind leukocytes to endothelial cells, an initial and essential step toward inflammation. In the present study, we used human umbilical vascular endothelial cells (HUVECs) to examine the effect of Ang1 on VEGF-induced expression of three adhesion molecules: intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. Interestingly, Ang1 suppressed VEGF-induced expression of these adhesion molecules. Furthermore, Ang1 reduced VEGF-induced leukocyte adhesion to HUVECs. These results demonstrate that Ang1 counteracts VEGF-induced inflammation by reducing VEGF-induced endothelial adhesiveness.
Impaired Cardiac Functional Reserve and Left Ventricular Hypertrophy in Adult Sheep After Prenatal Dexamethasone Exposure
Authors: Dodic Miodrag , Samuel Chrishan , Moritz Karen , Wintour E. Marelyn , Morgan John , Grigg Leeanne , Wong James
Published: September 2007

Abstract—

We have shown that exposure of pregnant ewes to dexamethasone (11.5 mg/d for 2 days) at 27 days of gestation (term, 150 days) led to increased blood pressure and cardiac output in adult offspring. In this study, we hypothesized that dexamethasone-induced hypertension is associated with left ventricular hypertrophy and a reduced cardiac functional reserve (CO
max-0
). Six control animals (group C) and five dexamethasone-exposed animals (group D) were volume-loaded with Hemaccel until the wedge pressure was 13 mm Hg (baseline). The wedge pressure was held constant during an infusion of dobutamine at incremental doses (0.4 to 12 μg/kg/min) while blood pressure and cardiac output were measured. The same protocol was repeated in each animal 5 days later under mild general anesthesia (1.5% isoflurane), when transthoracic echocardiography (M-mode) was obtained. Group D showed a reduced CO
max-0
in response to dobutamine during both conscious (89±22 versus 150±25 mL/kg/min in control;
P
<0.01) and anesthetized states (91±38 versus 156±56 mL/kg/min in control;
P
<0.05). Reduced CO
max-0
in group D was associated with higher left ventricular mass index compared with group C (2.6±0.67 versus 1.8±0.51 g/kg;
P
<0.05). In addition, group D showed a reduced cardiac contractility reserve (FS
max-0
) in response to dobutamine (21±22% versus 54±34% in group C;
P
<0.05). An impaired cardiac functional reserve in group D was associated with increased left ventricular type I collagen content. In conclusion, brief prenatal exposure to dexamethasone led to the development of hypertension, left ventricular hypertrophy, and reduced cardiac functional reserve in adult life.
Potentiation of Ca
2+
Release by cADP-Ribose in the Heart Is Mediated by Enhanced SR Ca
2+
Uptake Into the Sarcoplasmic Reticulum
Authors: Lukyanenko Valeriy , Györke Inna , Wiesner Theodore F. , Györke Sandor
Published: September 2007

Abstract—

cADP-Ribose (cADPR) is a novel endogenous messenger that is believed to mobilize Ca
2+
from ryanodine-sensitive Ca
2+
stores. Despite intense research, the precise mechanism of action of cADPR remains uncertain, and experimental findings are contradictory. To elucidate the mechanism of cADPR action, we performed confocal Ca
2+
imaging in saponin-permeabilized rat ventricular myocytes. Exposure of the cells to cADPR resulted in a slow (>2 minutes) and steady increase in the frequency of Ca
2+
sparks. These effects on local release events were accompanied by a significant increase in sarcoplasmic reticulum (SR) Ca
2+
content. In comparison, sensitization of ryanodine receptors (RyRs) by caffeine, a true RyR agonist, caused a rapid (<1 second) and transient potentiation of Ca
2+
sparks followed by a decrease in SR Ca
2+
content. When the increase in the SR load was prevented by partial inhibition of the SR Ca
2+
with thapsigargin, cADPR failed to produce any increase in sparking activity. cADPR had no significant impact on activity of single cardiac RyRs incorporated into lipid bilayers. However, it caused a significant increase in the rate of Ca
2+
uptake by cardiac SR microsomes. Our results suggest that the primary target of cADPR is the SR Ca
2+
uptake mechanism. Potentiation of Ca
2+
release by cADPR is mediated by increased accumulation of Ca
2+
in the SR and subsequent luminal Ca
2+
-dependent activation of RyRs.
Interleukin-18/Interleukin-18 Binding Protein Signaling Modulates Atherosclerotic Lesion Development and Stability
Authors: Mallat Ziad , Corbaz Anne , Scoazec Alexandra , Graber Pierre , Alouani Sami , Esposito Bruno , Humbert Yves , Chvatchko Yolande , Tedgui Alain
Published: September 2007

Abstract—

Interleukin (IL)-18 is the interferon-γ–inducing factor and has other proinflammatory properties. The precise role of IL-18 in immunoinflammatory diseases remains poorly understood. In this study, we show that in vivo electrotransfer of an expression-plasmid DNA encoding for murine IL-18 binding protein (BP) (the endogenous inhibitor of IL-18) prevents fatty streak development in the thoracic aorta of apoE knockout mice and slows progression of advanced atherosclerotic plaques in the aortic sinus. More importantly, transfection with the IL-18BP plasmid induces profound changes in plaque composition (decrease in macrophage, T cell, cell death, and lipid content and increase in smooth muscle cell and collagen content) leading to a stable plaque phenotype. These results identify for the first time a critical role for IL-18/IL-18BP regulation in atherosclerosis and suggest a potential role for IL-18 inhibitors in reduction of plaque development/progression and promotion of plaque stability. The full text of this article is available at http://www.circresaha.org.
Endothelial G Protein β-Subunits Trigger Nitric Oxide– but not Endothelium-Derived Hyperpolarizing Factor–Dependent Dilation in Rabbit Resistance Arteries
Authors: Véquaud Philippe , Thorin Eric
Published: September 2007

A single subtype of heterotrimeric G protein–coupled receptor controls both nitric oxide (NO) (sensitive to
l
-arginine analogues) and endothelium-derived hyperpolarizing factor (EDHF) (sensitive to high-external K
+
and apamine) production by the vascular endothelium leading to dilation. We hypothesized that α- and βγ-subunits of the G protein serve as distinct intermediates to produce NO and EDHF. In pressurized resistance arteries, selective pinocytotic endothelial incorporation of specific antibodies (Abs) directed against α
q/11
-subunits abolished acetylcholine (Ach)-mediated dilation but failed to influence oxymetazoline (Oxy, α
2
-adrenergic receptor agonist)-induced dilation. In contrast, α
i1-2
-subunit Abs prevented Oxy- but not Ach-induced dilation. Thus, as expected, endothelial muscarinic and α
2
-adrenoceptors couple to G
q
protein and G
i
proteins, respectively. β-subunit Abs reduced both Ach- and Oxy-induced dilation. The β-subunit Abs abolished the nitro-
l
-arginine (L-NNA)-sensitive component but did not impair the high-external K
+
-sensitive component of the dilation induced by Ach and Oxy. Thus, G protein β-subunits primarily accounted for NO production. Neutralization of Hsp90 and inhibition of the phospholipase C by U73122 (1 μmol/L) or intracellular Ca
2+
buffering with BAPTA-AM (10 μmol/L) sharply reduced NO-dependent but not K
+
-sensitive dilation. In conclusion, mobilization of the G protein β-subunit is pivotal to NO-dependent dilation triggered through muscarinic and α
2
-adrenergic receptors. In contrast, receptor-operated EDHF-dependent dilation was insensitive to β-subunit Abs. Although not directly activating the NO pathway, α-subunit activation is an absolute prerequisite for receptor-operated endothelium-dependent dilation of resistance arteries.
Catalytic Oligodeoxynucleotides Define a Key Regulatory Role for Early Growth Response Factor-1 in the Porcine Model of Coronary In-Stent Restenosis
Authors: Lowe Harry C. , Fahmy Roger G. , Kavurma Mary M. , Baker Andrew , Chesterman Colin N. , Khachigian Levon M.
Published: September 2007

Early growth response factor-1 (Egr-1) controls the expression of a growing number of genes involved in the pathogenesis of atherosclerosis and postangioplasty restenosis. Egr-1 is activated by diverse proatherogenic stimuli. As such, this transcription factor represents a key molecular target in efforts to control vascular lesion formation in humans. In this study, we have generated DNAzymes targeting specific sequences in human EGR-1 mRNA. These molecules cleave in vitro transcribed EGR-1 mRNA efficiently at preselected sites, inhibit EGR-1 protein expression in human aortic smooth muscle cells, block serum-inducible cell proliferation, and abrogate cellular regrowth after mechanical injury in vitro. These DNAzymes also selectively inhibit EGR-1 expression and proliferation of porcine arterial smooth muscle cells and reduce intimal thickening after stenting pig coronary arteries in vivo. These findings demonstrate that endoluminally delivered DNAzymes targeting EGR-1 may serve as inhibitors of in-stent restenosis.
Prostaglandin H Synthase and Vascular Function
Authors: Davidge Sandra T.
Published: September 2007

Prostaglandin H synthase (PGHS) is a rate-limiting enzyme in the production of prostaglandins and thromboxane, which are important regulators of vascular function. Under normal physiological conditions, PGHS-dependent vasodilators (such as prostacyclin) modulate vascular tone. However, PGHS-dependent vasoconstriction (mediated by thromboxane and/or its immediate precursor, PGH2) predominates in some vascular pathologies (eg, systemic hypertension, diabetes, cerebral ischemia, and aging). This review will discuss the role of PGHS-dependent modulation of vascular function in a number of vascular beds (systemic, pulmonary, cerebral, and uterine) with an emphasis on vascular pathophysiology. Moreover, the specific contributions of the different isoforms (PGHS-1 and PGHS-2) are discussed. Understanding the role of PGHS in vascular function is of particular importance because they are the targets of the commonly used nonsteroidal antiinflammatory drugs (NSAIDs), which include aspirin and ibuprofen. Importantly, with the advent of specific PGHS-2 inhibitors for treatment of conditions such as chronic inflammatory disease, it is an opportune time to review the data regarding PGHS-dependent modulation of vascular function.
ERK and p38 MAPK, but not NF-κB, Are Critically Involved in Reactive Oxygen Species–Mediated Induction of IL-6 by Angiotensin II in Cardiac Fibroblasts
Authors: Sano Motoaki , Fukuda Keiichi , Sato Toshihiko , Kawaguchi Haruko , Suematsu Makoto , Matsuda Satoshi , Koyasu Shigeo , Matsui Hideo , Yamauchi-Takihara Keiko , Harada Masaki , Saito Yoshihiko , Ogawa Satoshi
Published: September 2007

We recently reported that angiotensin II (Ang II) induced IL-6 mRNA expression in cardiac fibroblasts, which played an important role in Ang II–induced cardiac hypertrophy in paracrine fashion. The present study investigated the regulatory mechanism of Ang II–induced IL-6 gene expression, focusing especially on reactive oxygen species (ROS)-mediated signaling in cardiac fibroblasts. Ang II increased intracellular ROS in cardiac fibroblasts, and the increase was completely inhibited by the AT-1 blocker candesartan and the NADH/NADPH oxidase inhibitor diphenyleneiodonium (DPI). We first confirmed that antioxidant
N
-acetylcysteine, superoxide scavenger Tiron, and DPI suppressed Ang II–induced IL-6 expression. Because we observed that exogenous H
2
O
2
also increased IL-6 mRNA, the signaling pathways downstream of Ang II and exogenous H
2
O
2
were compared. Ang II, as well as exogenous H
2
O
2
, activated ERK, p38 MAPK, and JNK, which were significantly inhibited by
N
-acetylcysteine and DPI. In contrast with exogenous H
2
O
2
, however, Ang II did not influence phosphorylation and degradation of IκB-α/β or nuclear translocation of p65, nor did it increase NF-κB promoter activity. PD98059 and SB203580 inhibited Ang II–induced IL-6 expression. Truncation and mutational analysis of the IL-6 gene promoter showed that CRE was an important
cis
-element in Ang II–induced IL-6 gene expression. NF-κB–binding site was important for the basal expression of IL-6, but was not activated by Ang II. Ang II phosphorylated CREB through the ERK and p38 MAPK pathway in a ROS-sensitive manner. Collectively, these data indicated that Ang II stimulated ROS production via the AT1 receptor and NADH/NADPH oxidase, and that these ROS mediated activation of MAPKs, which culminated in IL-6 gene expression through a CRE-dependent, but not NF-κB–dependent, pathway in cardiac fibroblasts.
On the Role of Ca
2+
- and Voltage-Dependent Inactivation in Ca
v
1.2 Sensitivity for the Phenylalkylamine (-)Gallopamil
Authors: Sokolov Stanislav , Timin Eugen , Hering Steffen
Published: September 2007

Abstract—

L-type calcium channels (Ca
v
1.m) inactivate in response to elevation of intracellular Ca
2+
(Ca
2+
-dependent inactivation) and additionally by conformational changes induced by membrane depolarization (fast and slow voltage-dependent inactivation). Molecular determinants of inactivation play an essential role in channel inhibition by phenylalkylamines (PAAs). The relative impacts, however, of Ca
2+
-dependent and voltage-dependent inactivation in Ca
v
1.2 sensitivity for PAAs remain unknown. In order to analyze the role of the different inactivation processes, we expressed Ca
v
1.2 constructs composed of different β-subunits (β
1a
-, β
2a
-, or β
3
-subunit) in Xenopus oocytes and estimated their (-)gallopamil sensitivity by means of the two-microelectrode voltage clamp with either Ba
2+
or Ca
2+
as charge carrier. Ca
v
1.2 consisting of the β
2a
-subunit displayed the slowest inactivation and the lowest apparent sensitivity for the PAA (-)gallopamil. A significantly higher apparent (-)gallopamil-sensitivity with Ca
2+
as charge carrier was observed for all 3 β-subunit compositions. The kinetics of Ca
2+
-dependent inactivation and slow voltage-dependent inactivation were not affected by drug. The higher sensitivity of the Ca
v
1.2 channels for (-)gallopamil with Ca
2+
as charge carrier results from slower recovery (τ
rec,Ca
≈15 seconds versus τ
rec,Ba
≈3 to 5 seconds) from a PAA-induced channel conformation. We propose a model where (-)gallopamil promotes a fast voltage-dependent component in Ca
v
1.2 inactivation. The model reproduces the higher drug sensitivity in Ca
2+
as well as the lower sensitivity of slowly inactivating Ca
v
1.2 composed of the β
2a
-subunit.
Ischemic and Pharmacological Preconditioning in Girardi Cells and C2C12 Myotubes Induce Mitochondrial Uncoupling
Authors: Minners Jan , Lacerda Lydia , McCarthy Joy , Meiring James J. , Yellon Derek M. , Sack Michael N.
Published: September 2007

Pharmacological uncoupling of mitochondrial oxidation from phosphorylation promotes preconditioning-like cardioprotection in the isolated rat heart. We hypothesized that modest mitochondrial uncoupling may be a critical cellular event in orchestrating preconditioning. Human-derived Girardi cells and murine C2C12 skeletal myotubes were preconditioned using simulated ischemia, adenosine, and diazoxide. Cell viability after 6 hours of simulated ischemia was measured using lactate dehydrogenase release and propidium iodide uptake. Mitochondrial inner membrane potential (ΔΨm) was investigated by flow cytometry, cellular ATP by recombinant firefly-luciferase bioluminescence, and cellular oxygen consumption using oximetry. Preconditioning enhanced cell viability with attenuation of lactate dehydrogenase release (≥30%,
P
<0.05 versus ischemic controls) and a reduction in propidium iodide uptake by ≥26% versus ischemic controls after simulated ischemia in both cell lines. In Girardi cells, preconditioning induced the following phenotype immediately before index ischemia: (1) decreased ΔΨm (JC-1: simulated ischemia 90±3%, adenosine 82±7%, diazoxide 87±4%, versus control 100%,
P
<0.05); (2) attenuation in cellular ATP levels (CTL 0.21±0.03 nmol/L ATP/μg protein, simulated ischemia 0.12±0.02, adenosine 0.15±0.02, diazoxide 0.11±0.02,
P
<0.05); and (3) enhanced cellular oxygen consumption (control 2.3±0.1 nmol/L oxygen/min/1×10
6
cells, simulated ischemia 3.1±0.1, adenosine 3.1±0.3, diazoxide 2.6±0.2,
P
<0.05). Cytoprotection, mitochondrial depolarization, and enhanced oxygen consumption were attenuated by the putative mitochondrial K
ATP
-channel antagonist 5-hydroxydecanoate. The uncoupled phenotype in response to preconditioning was similarly observed in C2C12 myotubes. The present study suggests that modest mitochondrial uncoupling represents a unifying cellular response which may be important in directing preconditioning-mediated cytoprotection.
A Novel Mechanism for Myocardial Stunning Involving Impaired Ca
2+
Handling
Authors: Kim Song-Jung , Kudej Raymond K. , Yatani Atsuko , Kim Young-Kwon , Takagi Gen , Honda Ritsu , Colantonio David A. , Van Eyk Jennifer E. , Vatner Dorothy E. , Rasmusson Randall L. , Vatner Stephen F.
Published: September 2007

The mechanism of myocardial stunning has been studied extensively in rodents and is thought to involve a decrease in Ca
2+
responsiveness of the myofilaments, degradation of Troponin I (TnI), and no change in Ca
2+
handling. We studied the mechanism of stunning in isolated myocytes from chronically instrumented pigs. Myocytes were isolated from the ischemic (stunned) and nonischemic (normal) regions after 90-minute coronary stenosis followed by 60-minute reperfusion. Baseline myocyte contraction was reduced,
P
<0.01, in stunned myocytes (6.3±0.4%) compared with normal myocytes (8.8±0.4%). The time for 70% relaxation was prolonged,
P
<0.01, in stunned myocytes (131±8 ms) compared with normal myocytes (105±5 ms). The impaired contractile function was associated with decreased Ca
2+
transients (stunned, 0.33±0.04 versus normal, 0.49±0.05,
P
<0.01). Action potential measurements in stunned myocytes demonstrated a decrease in plateau potential without a change in resting membrane potential. These changes were associated with decreased L-type Ca
2+
-current density (stunned, −4.8±0.4 versus normal, −6.6±0.4 pA/pF,
P
<0.01). There were no differences in TnI, sarcoplasmic reticulum Ca
2+
ATPase (SERCA2a), and phospholamban protein quantities. However, the fraction of phosphorylated phospholamban monomer was reduced in stunned myocardium. In rats, stunned myocytes demonstrated reduced systolic contraction but actually accelerated relaxation and no change in Ca
2+
transients. Thus, mechanisms of stunning in the pig are radically different from the widely held concepts derived from studies in rodents and involve impaired Ca
2+
handling and dephosphorylation of phospholamban, but not TnI degradation.
Electrical Properties and Conduction in Reperfused Papillary Muscle
Authors: Cascio Wayne E. , Yang Hua , Johnson Timothy A. , Muller-Borer Barbara J. , Lemasters John J.
Published: September 2007

The reversibility of ischemia-induced changes of extracellular K
+
concentration ([K
+
]
o
), resting membrane potential (E
M
), and passive cable-like properties, ie, extracellular resistance and cell-to-cell electrical coupling, and their relationship to recovery of conduction and contraction is described in 25 reperfused rabbit papillary muscles. No-flow ischemia caused extracellular K
+
accumulation, depolarization of E
M
, an increase in whole-tissue (r
t
), external (r
o
), and internal (r
i
) longitudinal resistances, and failure of conduction and contraction. Muscles were reperfused 10 minutes after the onset of ischemia related cell-to-cell electrical uncoupling, ie, 26±1 minutes after arrest of perfusion. In 11 muscles, incomplete reflow occurred with only partial recovery of [K
+
]
o
and r
t
. In the remaining 14 muscles, reperfusion caused a rapid and parallel decrease in [K
+
]
o
, r
t
, and r
o
. When complete tissue reperfusion occurred, cell-to-cell electrical uncoupling was largely reversible. Thus, cell-to-cell electrical uncoupling did not indicate irreversible injury. Reperfusion induced a depolarizing current widening the difference between the K
+
equilibrium potential and the E
M
. This difference decreased after longer periods of reperfusion. Conduction was restored and conduction velocity approached preischemic values as cell-to-cell electrical interaction was reestablished and E
M
recovered. The recovery of r
o
preceded r
i
, decreasing the ratio of the extracellular to intracellular resistance early in reperfusion, an effect predicted to influence the amplitude of the extracellular voltage field and electrocardiographic ST segments during reperfusion.
Role of Ischemia and of Hypoxia-Inducible Genes in Arteriogenesis After Femoral Artery Occlusion in the Rabbit
Authors: Deindl Elisabeth , Buschmann Ivo , Hoefer Imo E. , Podzuweit Thomas , Boengler Kerstin , Vogel Sabina , van Royen Niels , Fernandez Borja , Schaper Wolfgang
Published: September 2007

Vascular endothelial growth factor (VEGF) is known to play an important role in angiogenesis. Its place in collateral artery growth (arteriogenesis), however, is still debated. In the present study, we analyzed the expression of VEGF and its receptors (Flk-1 and Flt-1) in a rabbit model of collateral artery growth after femoral artery occlusion. Hypoxia presents the most important stimulus for VEGF expression. We therefore also investigated the expression level of distinct hypoxia-inducible genes (HIF-1α, LDH A) and determined metabolic intermediates indicative for ischemia (ATP, creatine phosphate, and their catabolites). We found that arteriogenesis was not associated with an increased expression of VEGF or the mentioned hypoxia-inducible genes. Furthermore, the high-energy phosphates and their catabolites were entirely within normal limits. Despite the absence of an increased expression of VEGF and its receptors, collateral vessels increased their diameter by a factor of 10. The speed of collateral development could be increased by infusion of the chemoattractant monocyte chemotactic protein-1 but not by infusion of a 30 times higher concentration of VEGF. From these data, we conclude that under nonischemic conditions, arteriogenesis is neither associated with nor inducible by increased levels of VEGF and that VEGF is not a natural agent to induce arteriogenesis in vivo.
Antiischemic Effects of SB203580 Are Mediated Through the Inhibition of p38α Mitogen-Activated Protein Kinase
Authors: Martin Jody L. , Avkiran Metin , Quinlan Roy A. , Cohen Philip , Marber Michael S.
Published: September 2007

The aim of the present study was to determine whether the attenuation of myocardial ischemic injury by SB203580 is due to the inhibition of p38 mitogen-activated protein kinase (MAPK) or to other documented nonspecific effects of the drug. We made adenoviral vectors encoding the α isoform of p38 MAPK with or without site-directed mutations to prevent SB203580 binding and inhibition. In embryonal rat heart–derived cells and adult rat cardiocytes expressing wild-type p38α MAPK, injury was reduced significantly by SB203580 present during simulated ischemia. In contrast, SB203580 did not protect cells expressing the SB203580-resistant form of p38α MAPK. These observations suggest that SB203580-mediated protection depends on the inhibition of p38α MAPK.
Leukocyte Integrin Mac-1 Recruits Toll/Interleukin-1 Receptor Superfamily Signaling Intermediates to Modulate NF-κB Activity
Authors: Shi Can , Zhang Xiaobin , Chen Zhiping , Robinson Martyn K. , Simon Daniel I.
Published: September 2007

The leukocyte integrin Mac-1 (αMβ2, CD11b/CD18) regulates important cell functions in inflammation, including adhesion, phagocytosis, and oxidative burst. Deficiency of Mac-1 reduces vessel wall inflammation and neointimal thickening after murine carotid artery injury. Although Mac-1 has been implicated in modulating AP-1 and NF-κB activity, the signal transduction pathways involved are undefined. cDNA array analysis of Mac-1–clustered compared with –nonclustered monocytic THP-1 cells showed increased expression of the signal transducer TRAF6 (TNF receptor–associated factor 6), leading us to consider the possibility that Mac-1 used a Toll/IL-1 receptor family–like signaling pathway. Mac-1–dependent activation of NF-κB was potentiated by wild-type, and attenuated by dominant negative, TRAF6- and TGF-β–activated kinase (TAK1) constructs. IRAK1 (IL-1 receptor associated kinase), a kinase immediately upstream of TRAF6, coimmunoprecipitated with Mac-1. Taken together, these observations indicate that Mac-1 recruits a Toll/IL-1 receptor family–like cascade to modulate NF-κB activity. This represents a new pathway for integrin-dependent modulation of gene expression.
Inhibition of Transforming Growth Factor-β Signaling Accelerates Atherosclerosis and Induces an Unstable Plaque Phenotype in Mice
Authors: Mallat Ziad , Gojova Andrea , Marchiol-Fournigault Carmen , Esposito Bruno , Kamaté Caroline , Merval Régine , Fradelizi Didier , Tedgui Alain
Published: September 2007

Atherosclerosis is a disease of the arterial wall that seems to be tightly modulated by the local inflammatory balance. Whereas a large body of evidence supports a role for proinflammatory mediators in disease progression, the understanding of the role of the antiinflammatory component in the modulation of plaque progression is only at its beginning. TGF-β1, -β2, and -β3 are cytokines/growth factors with broad activities on cells and tissues in the cardiovascular system and have been proposed to play a role in the pathogenesis of atherosclerosis. However, no study has examined the direct role of TGF-β in the development and composition of advanced atherosclerotic lesions. In the present study, we show that inhibition of TGF-β signaling using a neutralizing anti–TGF-β1, -β2, and -β3 antibody accelerates the development of atherosclerotic lesions in apoE-deficient mice. Moreover, inhibition of TGF-β signaling favors the development of lesions with increased inflammatory component and decreased collagen content. These results identify a major protective role for TGF-β in atherosclerosis.
Differential Activation of Mitogen-Activated Protein Kinase Cascades and Apoptosis by Protein Kinase C ε and δ in Neonatal Rat Ventricular Myocytes
Authors: Heidkamp Maria C. , Bayer Allison L. , Martin Jody L. , Samarel Allen M.
Published: September 2007

Protein kinase C (PKC) ε and PKCδ translocation in neonatal rat ventricular myocytes (NRVMs) is accompanied by subsequent activation of the ERK, JNK, and p38
MAPK
cascades; however, it is not known if either or both novel PKCs are necessary for their downstream activation. Use of PKC inhibitors to answer this question is complicated by a lack of isoenzyme specificity, and the fact that many PKC inhibitors stimulate JNK and p38
MAPK
activity. Therefore, replication-defective adenoviruses (Advs) encoding constitutively active (ca) mutants of PKCε and PKCδ were used to test if either or both of these PKCs are sufficient to activate ERKs, JNKs, and/or p38
MAPK
in NRVMs. Adv-caPKCε infection (1 to 25 multiplicities of viral infection (MOI); 4 to 48 hours) increased total PKCε levels in a time- and dose-dependent manner, with maximal expression observed 8 hours after Adv infection. Adv-caPKCε induced a time- and dose-dependent increase in phosphorylated p42 and p44 ERKs, as compared with a control Adv encoding β-galactosidase (Adv-neβgal). Maximal ERK phosphorylation occurred 8 hours after Adv infection. In contrast, JNK was only minimally activated, and p38
MAPK
was relatively unaffected. Adv-caPKCδ infection (1 to 25 MOI, 4 to 48 hours) increased total PKCδ levels in a similar fashion. Adv-caPKCδ (5 MOI) induced a 29-fold increase in phosphorylated p54 JNK, and a 15-fold increase in phosphorylated p38
MAPK
24 hours after Adv infection. In contrast, p42 and p44 ERK were only minimally activated. Whereas neither Adv induced NRVM hypertrophy, Adv-caPKCδ, but not Adv-caPKCε, induced NRVM apoptosis. We conclude that the novel PKCs differentially regulate MAPK cascades and apoptosis in an isoenzyme-specific and time-dependent manner.
Platelet-Derived Growth Factor Promotes the Expression of Peroxisome Proliferator-Activated Receptor γ in Vascular Smooth Muscle Cells by a Phosphatidylinositol 3-Kinase/Akt Signaling Pathway
Authors: Fu Mingui , Zhu Xiaojun , Wang Qian , Zhang Jifeng , Song Qing , Zheng Hui , Ogawa Wataru , Du Jie , Chen Yuqing E.
Published: September 2007

Vascular diseases such as atherosclerosis are characterized by abnormal accumulation of vascular smooth muscle cells (VSMCs) within the intimal lining. The intimal VSMCs exhibit an increased expression of peroxisome proliferator-activated receptor γ (PPARγ), and the administration of pharmacological PPARγ agonists attenuates vascular lesion formation. The factors that regulate PPARγ expression in the vasculature are poorly defined. Here we report that platelet-derived growth factor (PDGF) upregulates PPARγ by the phosphatidylinositol 3-kinase (PI3-kinase)/Akt signaling pathway. Using Northern-blotting and Western-blotting analyses, we observed that the levels of PPARγ mRNA and protein were increased by 2- to 3.5-fold in human aortic smooth muscle cells (HASMCs) treated with PDGF (20 ng/mL). This was abolished by preincubation of HASMCs with a PI3-kinase inhibitor (LY294002, 50 μmol/L), and partially inhibited by a MEK1 inhibitor (U0126, 10 μmol/L), but not affected by a p38 kinase inhibitor (SB202190, 10 μmol/L). In addition, overexpression of the dominant-negative p85 subunit of PI3-kinase or Akt proteins blocked the PDGF-induced PPARγ expression. Taken together, our results suggest that PDGF induces PPARγ expression in VSMCs by a PI3-kinase/Akt signaling pathway. The characterization of factors and signaling pathways that modulate PPARγ expression in VSMCs may have important implications for understanding the pathogenesis of vascular diseases.
Regulation of Calcium Sparks and Spontaneous Transient Outward Currents by RyR3 in Arterial Vascular Smooth Muscle Cells
Authors: Löhn Matthias , Jessner Wolfgang , Fürstenau Michael , Wellner Maren , Sorrentino Vincenzo , Haller Hermann , Luft Friedrich C. , Gollasch Maik
Published: September 2007

Intracellular Ca
2+
levels control both contraction and relaxation in vascular smooth muscle cells (VSMCs). Ca
2+
-dependent relaxation is mediated by discretely localized Ca
2+
release events through ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). These local increases in Ca
2+
concentration, termed sparks, stimulate nearby Ca
2+
-activated K
+
(BK) channels causing BK currents (spontaneous transient outward currents or STOCs). STOCs are hyperpolarizing currents that oppose vasoconstriction. Several RyR isoforms are coexpressed in VSMCs; however, their role in Ca
2+
spark generation is unknown. To provide molecular information on RyR cluster function and assembly, we examined Ca
2+
sparks and STOCs in RyR3-deficient freshly isolated myocytes of resistance-sized cerebral arteries from knockout mice and compared them to Ca
2+
sparks in cells from wild-type mice. We used RT-PCR to identify RyR1, RyR2, and RyR3 mRNA in cerebral arteries. Ca
2+
sparks in RyR3-deficient cells were similar in peak amplitude (measured as F/F
0
), width at half-maximal amplitude, and duration compared with wild-type cell Ca
2+
sparks. However, the frequency of STOCs (between −60 mV and −20 mV) was significantly higher in RyR3-deficient cells than in wild-type cells. Ca
2+
sparks and STOCs in both RyR3-deficient and wild-type cells were inhibited by ryanodine (10 μmol/L), external Ca
2+
removal, and depletion of SR Ca
2+
stores by caffeine (1 mmol/L). Isolated, pressurized cerebral arteries of RyR3-deficient mice developed reduced myogenic tone. Our results suggest that RyR3 is part of the SR Ca
2+
spark release unit and plays a specific molecular role in the regulation of STOCs frequency in mouse cerebral artery VSMCs after decreased arterial tone.
Elevated Sympathetic Nervous Activity in Mice Deficient in αCGRP
Authors: Oh-hashi Yoshio , Shindo Takayuki , Kurihara Yukiko , Imai Tomihiko , Wang Yuhui , Morita Hiroyuki , Imai Yasushi , Kayaba Yuji , Nishimatsu Hiroaki , Suematsu Yoshihiro , Hirata Yasunobu , Yazaki Yoshio , Nagai Ryozo , Kuwaki Tomoyuki , Kurihara Hiroki
Published: September 2007

α-Calcitonin gene-related peptide (αCGRP) is a pleiotropic neuropeptide implicated in a variety of physiological processes. To better understand the biological functions of αCGRP, we developed an α
CGRP
-null mouse model using a gene targeting approach. Recordings of mean arterial pressure (MAP) and heart rate (HR) showed that basal MAP and HR were significantly higher in both anesthetized and conscious, unrestrained α
CGRP
-null mice than in corresponding wild-type mice. The elevated MAP in α
CGRP
-null mice was shown to be the result of elevated peripheral vascular resistance by α-adrenergic blockade with prazosin and by transthoracic echocardiogram, which revealed no significant differences between α
CGRP
-null and wild-type mice in the stroke volume, fractional shortening, and ejection fraction. Moreover, evaluation of autonomic nervous activity by measuring HR after pretreatment of atropine and/or atenolol and by analyzing arterial baroreceptor reflexes showed sympathetic nervous activity to be significantly elevated in α
CGRP
-null mice; elevated levels of urinary catecholamine metabolites and decreased HR variability in mutant mice were also consistent with that finding. These findings suggest that αCGRP contributes to the regulation of cardiovascular function through inhibitory modulation of sympathetic nervous activity.
Molecular Composition of 4-Aminopyridine-Sensitive Voltage-Gated K
+
Channels of Vascular Smooth Muscle
Authors: Thorneloe Kevin S. , Chen Tim T. , Kerr Paul M. , Grier Elizabeth F. , Horowitz Burton , Cole William C. , Walsh Michael P.
Published: September 2007

Voltage-gated K
+
channels (Kv) play a critical role in regulating arterial tone by modulating the membrane potential of vascular smooth muscle cells. Our previous work demonstrated that the dominant 4-aminopyridine (4-AP)-sensitive, delayed rectifier Kv current of rabbit portal vein (RPV) myocytes demonstrates similar 4-AP sensitivity and biophysical properties to Kv1α-containing channels. To identify the molecular constituents underlying the 4-AP-sensitive Kv current of vascular myocytes, we characterized the expression pattern of Kv1α subunits and their modulatory Kvβ subunits in RPV. The mRNAs encoding pore-forming subunits Kv1.2, Kv1.4, and Kv1.5 were detected by reverse transcriptase-polymerase chain reaction (RT-PCR), whereas Kv1.1, Kv1.3, and Kv1.6 transcripts were undetectable. Kvβ1.1, β1.2, β1.3, β2.1, and β2.2 messages were expressed, whereas Kvβ3.1 and β4 mRNAs were undetected by RT-PCR. Kv1.2, Kv1.4, Kv1.5, Kvβ1.2, β1.3, and β2.1 proteins were detected in RPV by Western blotting and/or immunocytochemistry of freshly isolated myocytes. We provide the first evidence, from coimmunoprecipitation studies, for the formation of heteromultimeric Kv channel complexes composed of Kv1.2, Kv1.5, and Kvβ1.2 subunits in vascular smooth muscle.