bluuebunny/crossref_metadata_2025 huggingface dataset:
Sarcoplasmic Reticulum Ca
2+
/Calmodulin-Dependent Protein Kinase Is Altered in Heart Failure
Authors: Netticadan Thomas , Temsah Rana M. , Kawabata Kenichi , Dhalla Naranjan S.
Published: June 2012

Abstract
—Although Ca
2+
/calmodulin-dependent protein kinase-II (CaMK) is known to phosphorylate different Ca
2+
cycling proteins in the cardiac sarcoplasmic reticulum (SR) and regulate its function, the status of CaMK in heart failure has not been investigated previously. In this study, we examined the hypothesis that changes in the CaMK-mediated phosphorylation of the SR Ca
2+
cycling proteins are associated with heart failure. For this purpose, heart failure in rats was induced by occluding the coronary artery for 8 weeks, and animals with >30% infarct of the left ventricle wall plus septum mass were used. Noninfarcted left ventricle was used for biochemical assessment; sham-operated animals served as control. A significant depression in SR Ca
2+
uptake and release activities was associated with a decrease in SR CaMK phosphorylation of the SR proteins, ryanodine receptor (RyR), Ca
2+
pump ATPase (SR/endoplasmic reticulum Ca
2+
ATPase [SERCA2a]), and phospholamban (PLB) in the failing heart. The SR protein contents for RyR, SERCA2a, and PLB were decreased in the failing hearts. Although the SR Ca
2+
/calmodulin-dependent CaMK activity, CaMK content, and CaMK autophosphorylation were depressed, the SR phosphatase activity was enhanced in the failing heart. On the other hand, the cAMP-dependent protein kinase–mediated phosphorylation of RyR and PLB was not affected in the failing heart. On the basis of these results, we conclude that alterations in SR CaMK-mediated phosphorylation may be partly responsible for impaired SR function in heart failure.
Prospects for Gene Therapy for Heart Failure
Authors: Hajjar Roger J. , del Monte Federica , Matsui Takashi , Rosenzweig Anthony
Published: June 2012

Abstract
—Heart failure represents an enormous clinical challenge in need of effective therapeutic approaches. The possibility of gene therapy for heart failure merits consideration at this time because of improvements in vector technology; cardiac gene delivery; and, most importantly, our understanding of the molecular pathogenesis of heart failure. We will first review recent advances in cardiac gene delivery in animal models and then examine several targets being considered for therapeutic intervention. In this context, gene transfer provides not only a potential therapeutic modality but also an important tool to help validate specific targets. Several interventions, particularly those enhancing sarcoplasmic calcium transport, show promise in animal models of heart failure and in myopathic cardiomyocytes derived from patients. However, bridging the gap between these basic investigative studies and clinical gene therapy remains a formidable task. Early experiments in rodents will need to be extended to large-animal models with clinical-grade vectors and delivery systems to assess both efficacy and safety. On the basis of a foundation of rigorous science and a growing understanding of heart failure pathogenesis, there is reason for cautious optimism for the future.
A Mechanism of Transition From Ventricular Fibrillation to Tachycardia
Authors: Samie Faramarz H. , Mandapati Ravi , Gray Richard A. , Watanabe Yoshio , Zuur Charlotte , Beaumont Jacques , Jalife José
Published: June 2012

Abstract
—Abbreviation of the action potential duration and/or effective refractory period (ERP) is thought to decrease the cycle length of reentrant arrhythmias. Verapamil, however, paradoxically converts ventricular fibrillation (VF) to ventricular tachycardia (VT), despite reducing the ERP. This mechanism remains unclear. We hypothesize that the size and the dynamics of the core of rotating waves, in addition to the ERP, influence the arrhythmia manifestation (ie, VF or VT). The objectives of this study were (1) to demonstrate functional reentry as a mechanism of VF and VT in the isolated Langendorff-perfused rabbit heart in the absence of an electromechanical uncoupler and (2) to elucidate the mechanism of verapamil-induced conversion of VF to VT. We used high-resolution video imaging with a fluorescent dye, ECG, frequency and 2-dimensional phase analysis, and computer simulations. Activation patterns in 10 hearts were studied during control, verapamil perfusion (2×10
−6
mol/L), and washout. The dominant frequency of VF decreased from 16.2±0.7 to 13.5±0.6 Hz at 20 minutes of verapamil perfusion (
P
<0.007). Concomitantly, phase analysis revealed that wavefront fragmentation was reduced, as demonstrated by a 3-fold reduction in the density of phase singularities (PSs) on the ventricular epicardial surface (PS density: control, 1.04±0.12 PSs/cm
2
; verapamil, 0.32±0.06 PSs/cm
2
[
P
=0.0008]). On washout, the dominant frequency and the PS density increased, and the arrhythmia reverted to VF. The core area of transiently appearing rotors significantly increased during verapamil perfusion (control, 4.5±0.6 mm
2
; verapamil, 9.2±0.5 mm
2
[
P
=0.0002]). In computer simulations, blockade of slow inward current also caused an increase in the core size. Rotating waves underlie VF and VT in the isolated rabbit heart. Verapamil-induced VF-to-VT conversion is most likely due to a reduction in the frequency of rotors and a decrease in wavefront fragmentation that lessens fibrillatory propagation away from the rotor.
The Third Cytoplasmic Loop of the Angiotensin II Type 1 Receptor Exerts Differential Effects on Extracellular Signal–Regulated Kinase (ERK1/ERK2) and Apoptosis via Ras- and Rap1-Dependent Pathways
Authors: Haendeler Judith , Ishida Mari , Hunyady Laszlo , Berk Bradford C.
Published: June 2012

Abstract
—The third cytoplasmic loop of the angiotensin (Ang) II type 1 receptor (AT
1
) is important for receptor coupling to G proteins and activation of downstream events. Therefore, we determined whether specific AT
1
sequences were required for kinase activation and inhibition of apoptosis by transfecting wild-type (AT1Rwt) and mutated AT
1
into 293 cells. Ang II stimulated a 19.4-fold increase in extracellular signal–regulated kinase (ERK1/ERK2) activity in 293 cells transfected with AT1Rwt. However, in 293 cells that expressed a receptor in which amino acids 221 and 222 were deleted (AT1R[Del221/222]), Ang II–mediated ERK1/ERK2 activation was inhibited by >85%. In contrast, c-Jun NH
2
-terminal protein kinase (JNK) activation was similar in AT1Rwt- and AT1R(Del221/222)-transfected cells. Activation of ERK1/ERK2 by AT1Rwt was independent of Ca
2+
, whereas the low level of ERK1/ERK2 activation by AT1R(Del221/222) was completely Ca
2+
dependent. Activation of ERK1/ERK2 in AT1Rwt required Ras, whereas AT1R(Del221/222) required Rap1. These results demonstrate the presence of 2 different pathways for ERK1/ERK2 activation by Ang II, which differ in their requirements for Ca
2+
and small G proteins (Ras versus Rap1). Furthermore, Ang II prevented serum deprivation–induced apoptosis in cells transfected with AT1Rwt but not AT1R(Del221/222). AKT was only phosphorylated by Ang II in AT1Rwt-transfected cells. Overexpression of constitutively active AKT significantly reduced serum deprivation–induced apoptosis in cells transfected with AT1R(Del221/222). This study shows for the first time a direct link between kinase activation and inhibition of apoptosis dependent on amino acids 221 and 222 in the third cytoplasmic loop of the AT
1
.
Direct Visualization of Lipid Deposition and Reverse Lipid Transport in a Perfused Artery
Authors: Rutledge J. C. , Mullick A. E. , Gardner G. , Goldberg I. J.
Published: June 2012

Abstract
—The major goal of this study was to determine the interactions of VLDL surface and core lipids with the artery wall. We first demonstrated in vitro that surface lipid in VLDL could be traced using the phospholipid-like fluorescent probe 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine (DiI). The core of VLDL particles was traced by fluorescently labeling apolipoprotein B with TRITC. The labeled VLDLs were perfused through rat carotid arteries, and accumulation of the fluorescently labeled VLDL components in the arterial walls was determined by quantitative fluorescence microscopy. Addition of lipoprotein lipase increased the accumulation of both DiI and TRITC by >2.3-fold. Histological examination showed that DiI and TRITC were primarily localized to the endothelial layer; however, DiI also accumulated as small “lakes” deeper in the artery, in a subendothelial position. Addition of HDL to the perfusion decreased the accumulation of surface lipid and apolipoprotein B–containing particles and eliminated the DiI lakes. Moreover, the increase in endothelial layer permeability associated with lipolysis was attenuated 21% by HDL. If VLDL surface lipid first was allowed to accumulate in the arterial wall, its subsequent rate of loss was more than twice as fast if HDL was included in the perfusate. These studies directly demonstrate atherogenic effects of VLDL lipolysis and their inhibition by HDL.
Enhanced Cardiac Function in Transgenic Mice Expressing a Ca
2+
-Stimulated Adenylyl Cyclase
Authors: Lipskaia Larissa , Defer Nicole , Esposito Giovanni , Hajar Iman , Garel Marie-Claude , Rockman Howard A. , Hanoune Jacques
Published: June 2012

Abstract
—The predominant functional adenylyl cyclases normally expressed in cardiac tissue and coupled to β-adrenergic receptors are inhibited by micromolar Ca
2+
concentration. To modify the overall balance of activities, we have generated transgenic mice expressing the Ca
2+
-stimulatable adenylyl cyclase type 8 (AC8) specifically in the heart. AC activity is increased by at least 7-fold in heart membranes from transgenic animals and is stimulated by Ca
2+
in the same range of concentration that inhibits the endogenous activity. Moreover, the in vivo basal protein kinase A activity was augmented 4-fold. Overexpression of AC8 in the heart has no detrimental consequences on global cardiac function. Basal heart rate and contractile function, measured by noninvasive echocardiography, were unchanged. In contrast, on release of parasympathetic tone, the intrinsic contractility is heightened and unresponsive to further β-adrenergic receptor stimulation. AC8 transgenic mice thus represent an original model to investigate the relative influence of Ca
2+
and cAMP on cardiac function within a phenotype of enhanced cardiac contractility and relaxation.
β-Blockade Prevents Sustained Metalloproteinase Activation and Diastolic Stiffening Induced by Angiotensin II Combined With Evolving Cardiac Dysfunction
Authors: Senzaki Hideaki , Paolocci Nazareno , Gluzband Yehezkiel A. , Lindsey Merry L. , Janicki Joseph S. , Crow Michael T. , Kass David A.
Published: June 2012

Abstract
—Angiotensin II (Ang II)–mediated sympathostimulation may worsen the progression of cardiac failure, although the nature and mechanisms of such interactions are largely unknown. We previously demonstrated that Ang II combined with evolving cardiodepression (48-hour tachycardia pacing, 48hP) induces marked chamber stiffening and increases metalloproteinases (MMPs). Here, we test the hypothesis that both abnormalities stem from sympathostimulatory effects of Ang II. Forty-eight dogs were instrumented to serially assess conscious ventricular mechanics, MMP abundance and activity, and myocardial histopathology. 48hP combined with 5 days of Ang II (15±5 ng · kg
−1
· min
−1
IV) more than doubled chamber stiffness (end-diastolic pressure >25 mm Hg,
P
<0.001), whereas stiffness was unchanged by Ang II or 48hP alone. In vitro and in situ zymography revealed increased MMP abundance and activity (principally 92-kDa gelatinase) from Ang II+48hP. Both stiffening and MMP changes were prevented by cotreatment with high-dose atenolol (which nearly fully inhibited isoproterenol-induced inotropy) but not partial β-blockade. Myocellular damage with fibroblast/neutrophil infiltration from Ang II+48hP was also inhibited by high- but not low-dose atenolol, whereas collagen content was not elevated with either dose. These data support a role of sympathostimulation by Ang II in modulating myocardial MMP abundance and activity and diastolic stiffening in evolving heart failure and suggest a novel mechanism by which β-blockade may limit chamber remodeling and diastolic dysfunction.
cAMP-Dependent Vasodilators Cross-Activate the cGMP-Dependent Protein Kinase to Stimulate BK
Ca
Channel Activity in Coronary Artery Smooth Muscle Cells
Authors: White Richard E. , Kryman Jonathan P. , El-Mowafy Abdalla M. , Han Guichun , Carrier Gerald O.
Published: June 2012

Abstract
—cAMP-dependent vasodilators are used to treat a variety of cardiovascular disorders; however, the signal transduction pathways and effector mechanisms stimulated by these agents are not fully understood. In the present study we demonstrate that cAMP-stimulating agents enhance the activity of the large-conductance, calcium-activated potassium (BK
Ca
) channel in single myocytes from coronary arteries by “cross-activation” of the cGMP-dependent protein kinase (protein kinase G, PKG). Single-channel patch-clamp data revealed that 10 μmol/L isoproterenol, forskolin, or dopamine opens BK
Ca
channels in coronary myocytes and that this effect is attenuated by inhibitors of PKG (KT5823; Rp-8-pCPT-cGMPS), but not by inhibiting the cAMP-dependent protein kinase (protein kinase A, PKA). In addition, a membrane-permeable analog, CPT-cAMP, also opened BK
Ca
channels in these myocytes, and this effect was reversed by KT5823. Direct biochemical measurement confirmed that dopamine or forskolin stimulates PKG activity in coronary arteries but does not elevate cGMP. Finally, the stimulatory effect of cAMP on BK
Ca
channels was reconstituted in a cell-free, inside-out patch by addition of purified PKG activated by either cGMP or cAMP. In contrast, channel gating was unaffected by exposure to the purified catalytic subunit of PKA. In summary, findings from on-cell and cell-free patch-clamp experiments provide direct evidence that cAMP-dependent vasodilators open BK
Ca
channels in coronary myocytes by cross-activation of PKG (but not via PKA). Biochemical assay confirmed this cross-activation mechanism of cAMP action in these arteries. This signaling pathway is a novel mechanism for regulation of potassium channel activity in vascular smooth muscle and other cells.
Adenosine A
1
Receptor Induced Delayed Preconditioning in Rabbits
Authors: Dana Ali , Skarli Maria , Papakrivopoulou Jenny , Yellon Derek M.
Published: June 2012

Abstract
—Transient adenosine A
1
receptor (A
1
R) activation in rabbits induces delayed preconditioning against myocardial infarction 24 to 72 hours later. The cellular mechanisms downstream of A
1
R mediating this delayed cardioprotection have not been elucidated. This study examined the role of protein kinase C (PKC) and tyrosine kinases (TKs) in the signaling cascade mediating A
1
R-induced late preconditioning in rabbits. The small heat shock protein Hsp27 has been shown to confer cytoskeletal protection when in the phosphorylated state. We therefore also evaluated the potential role of the p38 mitogen-activated protein kinase (p38 MAPK) and Hsp27 as distal mediators of A
1
R-induced delayed preconditioning. Pharmacological preconditioning of rabbits with the selective A
1
agonist 2-chloro-
N
6
-cyclopentyladenosine (CCPA; 100 μg/kg) significantly reduced myocardial infarct size compared with control animals, after 30-minute regional ischemia/2-hour reperfusion in vivo 24 hours later (23.7±3.1 versus 43.0±4.1%;
P
<0.05). This delayed protection was abrogated by prior inhibition of either PKC with chelerythrine chloride (5 mg/kg) or of TKs with lavendustin A (1.3 mg/kg), suggesting that both PKC and TK are crucial for the development of delayed preconditioning after A
1
receptor activation in the rabbit. Myocardial tissue extracts obtained 24 hours after CCPA treatment were analyzed for p38 MAPK catalytic activity using an in vitro kinase assay. This showed an almost 7-fold increase in p38 MAPK activity in myocardial samples pretreated with CCPA compared with control hearts. Two-dimensional gel electrophoresis revealed an increase in the phosphorylated isoforms of Hsp27 in hearts pretreated with CCPA compared with control hearts. Prior inhibition of either PKC or TK prevented the CCPA-induced increase in p38 MAPK activity and phosphorylation of Hsp27. This study identifies new components of the signaling mechanism of A
1
R-induced delayed preconditioning. Our results suggest an important role for both PKC and TK as mediators of late preconditioning against infarction after A
1
R activation and, although correlative, point to the p38 MAPK/Hsp27 pathway as a potential distal effector of this protection.
Role of the JAK/STAT Pathway in Rat Carotid Artery Remodeling After Vascular Injury
Authors: Seki Yukihiko , Kai Hisashi , Shibata Rei , Nagata Tsuyoshi , Yasukawa Hideo , Yoshimura Akihiko , Imaizumi Tsutomu
Published: June 2012

Abstract
—In cultured vascular smooth muscle cells (VSMCs), Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) are expressed constitutively and play a role in angiotensin II (Ang II)–induced intracellular signaling and proliferation. However, little is known regarding the relevance of these proteins to the process of vascular remodeling. The role of JAK and STAT proteins in vascular remodeling and their functional coupling with Ang II were examined in balloon-injured rat carotid artery. Immunoreactive Jak2, Tyk2, Stat1, and Stat3 were not detected in the intact artery. Immunohistostaining showed transient expressions of these JAKs and STATs in medial and neointimal VSMCs at days 2 and 5, respectively, with a peak at day 7 in both layers. The expressions declined to insignificant levels by day 14. Ang II type 1 receptors (AT
1
s) were coexpressed in the medial and neointimal VSMCs expressing Jak2 and Stat3. The Jak2 and Stat3 inductions in the injured artery were accompanied by constitutive Jak2 and Stat3 phosphorylations, which were enhanced by ex vivo Ang II stimulation via AT
1
. Additionally, a Jak2 inhibitor, AG490, blocked the Ang II–induced Stat3 phosphorylation. Furthermore, local treatment with AG490 inhibited constitutive Stat3 phosphorylation and neointimal VSMC replication and subsequently reduced neointima formation in the injured artery. In conclusion, JAK and STAT proteins were inducible in medial and neointimal VSMCs after vascular injury and were functionally coupled to AT
1
. The inductions of JAKs and STATs would be involved in the mechanisms of neointima formation after vascular injury. (

Circ Res. 2000;87:12-18.)
A gp91phox Containing NADPH Oxidase Selectively Expressed in Endothelial Cells Is a Major Source of Oxygen Radical Generation in the Arterial Wall
Authors: Görlach A. , Brandes R. P. , Nguyen K. , Amidi M. , Dehghani F. , Busse R.
Published: June 2012

Abstract
—Reactive oxygen species (ROS) play an important role in regulating vascular tone and intracellular signaling; the enzymes producing ROS in the vascular wall are, however, poorly characterized. We investigated whether a functionally active NADPH oxidase similar to the leukocyte enzyme, ie, containing the subunits p22phox and gp91phox, is expressed in endothelial cells (ECs) and smooth muscle cells (SMCs). Phorbol 12-myristate 13-acetate (PMA), a stimulus for leukocyte NADPH oxidase, increased ROS generation in cultured ECs and endothelium-intact rat aortic segments, but not in SMCs or endothelium-denuded arteries. NADPH enhanced chemiluminescence in all preparations. p22phox mRNA and protein was detected in ECs and SMCs, whereas the expression of gp91phox was confined to ECs. Endothelial gp91phox was identical to the leukocyte form as determined by sequence analysis. In contrast, mitogenic oxidase-1 (mox1) was expressed in SMCs, but not in ECs. To determine the functional relevance of gp91phox expression, experiments were performed in aortic segments from wild-type, gp91phox
−
/−
, and endothelial NO synthase (eNOS)
−
/−
mice. PMA-induced ROS generation was comparable in aortae from wild-type and eNOS
−
/−
mice, but was attenuated in segments from gp91phox
−
/−
mice. Endothelium-dependent relaxation was greater in aortae from gp91phox
−
/−
than from wild-type mice. The ROS scavenger tiron increased endothelium-dependent relaxation in segments from wild-type, but not from gp91phox
−
/−
mice. These data demonstrate that ECs, in contrast to SMCs, express a gp91phox-containing leukocyte-type NADPH oxidase. This enzyme is a major source for arterial ROS generation and affects the bioavailability of endothelium-derived NO. (

Circ Res. 2000;87:26-32.)
Homocysteine Thiolactone and Protein Homocysteinylation in Human Endothelial Cells
Authors: Jakubowski Hieronim , Zhang Li , Bardeguez Arlene , Aviv Abram
Published: June 2012

Abstract
—Editing of the nonprotein amino acid homocysteine by certain aminoacyl-tRNA synthetases results in the formation of the thioester homocysteine thiolactone. Here we show that in the presence of physiological concentrations of homocysteine, methionine, and folic acid, human umbilical vein endothelial cells efficiently convert homocysteine to thiolactone. The extent of this conversion is directly proportional to homocysteine concentration and inversely proportional to methionine concentration, suggesting involvement of methionyl-tRNA synthetase. Folic acid inhibits the synthesis of thiolactone by lowering homocysteine and increasing methionine concentrations in endothelial cells. We also show that the extent of post-translational protein homocysteinylation increases with increasing homocysteine levels but decreases with increasing folic acid and HDL levels in endothelial cell cultures. These data support a hypothesis that metabolic conversion of homocysteine to thiolactone and protein homocysteinylation by thiolactone may play a role in homocysteine-induced vascular damage. (

Circ Res. 2000;87:45-51.)
Lysophosphatidylcholine Activates p38 and p42/44 Mitogen-Activated Protein Kinases in Monocytic THP-1 Cells, but Only p38 Activation Is Involved in Its Stimulated Chemotaxis
Authors: Jing Qing , Xin Sun-Mei , Zhang Wen-Bo , Wang Ping , Qin Yong-Wen , Pei Gang
Published: June 2012

Abstract
—Oxidized LDLs (OxLDLs) have been shown to be involved in recruitment of blood monocytes into the arterial subendothelial space, which is the earliest step in atherogenesis, but the underlying molecular mechanisms are poorly understood. The present study demonstrated that lysophosphatidylcholine (LPC), a major phospholipid component of OxLDL, strongly evoked phosphorylation and activation of p38 and p42/44 mitogen-activated protein kinases in monocytic cells. The stimulation of p38 and p42/44 occurred in a dose- and time-dependent manner, reaching the maximal activation at 25 μg/mL LPC within 5 minutes. Interestingly, inhibition of p38 activation by OxLDL or LPC, using its selective inhibitors (SB203580 and
SKF86002
), completely blocked OxLDL- or LPC-stimulated chemotaxis of THP-1 cells, which was measured in a transwell chemotaxis assay. In contrast, inhibition of p42/44 activation by its potent inhibitor (PD98059) did not block OxLDL- or LPC-stimulated chemotaxis. Moreover, expression of a p38 dominant-negative mutant (p38AF) reduced cell chemotaxis significantly. In addition, activation of p38 by LPC was apparently mediated neither by scavenger receptors nor by tyrosine kinase receptors. It was, however, effectively blocked by pertussis toxin and substantially reduced by phospholipase C inhibitor (U73122) and phosphatidylinositol 3-kinase inhibitors (wortmannin and LY294002). LPC also inhibited forskolin-stimulated cAMP accumulation in a pertussis toxin–sensitive manner, indicating that Gi/Go proteins likely mediated the effects of LPC. Our results suggested that OxLDL/LPC efficiently activated both p38 and p42/44, but only the activation of p38 was functionally associated with OxLDL-/LPC-induced chemotaxis in THP-1 cells. (

Circ Res. 2000;87:52-59.)
Production of 20-HETE and Its Role in Autoregulation of Cerebral Blood Flow
Authors: Gebremedhin Debebe , Lange Andrew R. , Lowry Timothy F. , Taheri M. Reza , Birks Eric K. , Hudetz Antal G. , Narayanan Jayashree , Falck John R. , Okamoto Hirotsugu , Roman Richard J. , Nithipatikom Kasem , Campbell William B. , Harder David R.
Published: June 2012

Abstract
—In the brain, pressure-induced myogenic constriction of cerebral arteriolar muscle contributes to autoregulation of cerebral blood flow (CBF). This study examined the role of 20-HETE in autoregulation of CBF in anesthetized rats. The expression of P-450 4A protein and mRNA was localized in isolated cerebral arteriolar muscle of rat by immunocytochemistry and in situ hybridization. The results of reverse transcriptase–polymerase chain reaction studies revealed that rat cerebral microvessels express cytochrome P-450 4A1, 4A2, 4A3, and 4A8 isoforms, some of which catalyze the formation of 20-HETE from arachidonic acid. Cerebral arterial microsomes incubated with [
14
C]arachidonic acid produced 20-HETE. An elevation in transmural pressure from 20 to 140 mm Hg increased 20-HETE concentration by 6-fold in cerebral arteries as measured by gas chromatography/mass spectrometry. In vivo, inhibition of vascular 20-HETE formation with
N
-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), or its vasoconstrictor actions using 15-HETE or 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE), attenuated autoregulation of CBF to elevations of arterial pressure. In vitro application of DDMS, 15-HETE, or 20-HEDE eliminated pressure-induced constriction of rat middle cerebral arteries, and 20-HEDE and 15-HETE blocked the vasoconstriction action of 20-HETE. Taken together, these data suggest an important role for 20-HETE in the autoregulation of CBF. (

Circ Res. 2000;87:60-65.)
Distribution of a Persistent Sodium Current Across the Ventricular Wall in Guinea Pigs
Authors: Sakmann Bernhard F. A. S. , Spindler Anthony J. , Bryant Simon M. , Linz Klaus W. , Noble Denis
Published: June 2012

Abstract
—A tetrodotoxin-sensitive persistent sodium current,
I
pNa
, was found in guinea pig ventricular myocytes by whole-cell patch clamping. This current was characterized in cells derived from the basal left ventricular subendocardium, midmyocardium, and subepicardium. Midmyocardial cells show a statistically significant (
P
<0.05) smaller
I
pNa
than subendocardial and subepicardial myocytes. There was no significant difference in
I
pNa
current density between subepicardial and subendocardial cells. Computer modeling studies support a role of this current in the dispersion of action potential duration across the ventricular wall.
Increased Mitochondrial K
ATP
Channel Activity During Chronic Myocardial Hypoxia
Authors: Eells Janis T. , Henry Michele M. , Gross Garrett J. , Baker John E.
Published: June 2012

Abstract
—Increased resistance to myocardial ischemia in chronically hypoxic immature rabbit hearts is associated with activation of ATP-sensitive K
+
(K
ATP
) channels. We determined whether chronic hypoxia from birth alters the function of the mitochondrial K
ATP
channel. The K
ATP
channel opener bimakalim (1 μmol/L) increased postischemic recovery of left ventricular developed pressure in isolated normoxic (F
io
2
=0.21) hearts to values (42±4% to 67±5% ) not different from those of hypoxic controls but did not alter postischemic recovery of developed pressure in isolated chronically hypoxic (F
io
2
=0.12) hearts (69±5% to 72±5%). Conversely, the K
ATP
channel blockers glibenclamide (1 μmol/L) and 5-hydroxydecanoate (5-HD, 300 μmol/L) attenuated the cardioprotective effect of hypoxia but had no effect on postischemic recovery of function in normoxic hearts. ATP synthesis rates in hypoxic heart mitochondria (3.92±0.23 μmol ATP · min
−1
· mg mitochondrial protein
−1
) were significantly greater than rates in normoxic hearts (2.95±0.08 μmol ATP · min
−1
· mg mitochondrial protein
−1
). Bimakalim (1 μmol/L) decreased the rate of ATP synthesis in normoxic heart mitochondria consistent with mitochondrial K
ATP
channel activation and mitochondrial depolarization. The effect of bimakalim on ATP synthesis was antagonized by the K
ATP
channel blockers glibenclamide (1 μmol/L) and 5-HD (300 μmol/L) in normoxic heart mitochondria, whereas glibenclamide and 5-HD alone had no effect. In hypoxic heart mitochondria, the rate of ATP synthesis was not affected by bimakalim but was attenuated by glibenclamide and 5-HD. We conclude that mitochondrial K
ATP
channels are activated in chronically hypoxic rabbit hearts and implicate activation of this channel in the improved mitochondrial bioenergetics and cardioprotection observed.
Integrated Ca
2+
Signaling Between Smooth Muscle and Endothelium of Resistance Vessels
Authors: Yashiro Yasuaki , Duling Brian R.
Published: June 2012

Abstract
—Cell-cell communication in the arteriolar wall was examined using the Ca
2+
-sensitive indicator fura-2 and the Ca
2+
buffer BAPTA as means of measuring and buffering cellular Ca
2+
. The experiments focused on the role of endothelial cell [Ca
2+
]
i
in modulating phenylephrine (PE)-induced contractions in in vitro arterioles of the hamster cremaster. Fura-2-AM and BAPTA-AM were applied intraluminally to accomplish endothelium-specific loading. PE was applied to short segments of arterioles using pressure-pulse ejection from a micropipette. Under control conditions at the site of stimulation, PE elicited a strong vasoconstriction preceded by an increase in endothelial cell [Ca
2+
]
i
. A very small biphasic conducted response was observed at sites upstream from the stimulation site. BAPTA sharply reduced the measured Ca
2+
response in the endothelium. This was associated with an enhanced local contractile response. In addition, the biphasic conducted response was converted into a strong conducted vasoconstriction. PE caused an initial rise in smooth muscle [Ca
2+
]
i
at the stimulated site, which was followed by a rapid decrease below baseline. Endothelial cell loading of BAPTA had minimal effect on the initial [Ca
2+
]
i
peak but eliminated the secondary decrease in smooth muscle [Ca
2+
]
i
. Intraluminal application of charybdotoxin plus apamin mimicked the change in vasomotor state induced by BAPTA. These data lead us to hypothesize that, after smooth muscle stimulation, intercellular Ca
2+
signaling between smooth muscle and endothelium causes a secondary rise in endothelial cell Ca
2+
, which triggers a hyperpolarizing event and initiates a conducted vasodilation. We conclude that smooth muscle and endothelium operate as a functional unit in these vessels.
Epicardial Outgrowth Inhibition Leads to Compensatory Mesothelial Outflow Tract Collar and Abnormal Cardiac Septation and Coronary Formation
Authors: Gittenberger-de Groot Adriana C. , Vrancken Peeters Mark-Paul F.M. , Bergwerff Maarten , Mentink Monica M.T. , Poelmann Robert E.
Published: June 2012

In the present study, we investigated the modulatory role of the epicardium in myocardial and coronary development. Epicardial cell tracing experiments have shown that epicardium-derived cells are the source of interstitial myocardial fibroblasts, cushion mesenchyme, and smooth muscle cells. Epicardial outgrowth inhibition studies show abnormalities of the compact myocardial layer, myocardialization of cushion tissue, looping, septation, and coronary vascular formation. Lack of epicardial spreading is partly compensated by mesothelial outgrowth over the conotruncal region. Heterospecific epicardial transplant is able to partially rescue the myocardial development, as well as septation and coronary formation.
Vasodilator-Stimulated Phosphoprotein Serine 239 Phosphorylation as a Sensitive Monitor of Defective Nitric Oxide/cGMP Signaling and Endothelial Dysfunction
Authors: Oelze Matthias , Mollnau Hanke , Hoffmann Nina , Warnholtz Ascan , Bodenschatz Martin , Smolenski Albert , Walter Ulrich , Skatchkov Mikhail , Meinertz Thomas , Münzel Thomas
Published: June 2012

Abstract
—Studies with cGMP-dependent protein kinase I (cGK-I)-deficient human cells and mice demonstrated that cGK-I ablation completely disrupts the NO/cGMP pathway in vascular tissue, which indicates a key role of this protein kinase as a mediator of the NO/cGMP action. Analysis of the vasodilator-stimulated phosphoprotein phosphorylated at serine 239 (P-VASP) is a useful tool to monitor cGK-I activation in platelets and cultured endothelial and smooth muscle cells. Therefore, we investigated whether endothelial dysfunction and/or vascular NO bioavailability is reflected by decreased vessel wall P-VASP and whether improvement of endothelial dysfunction restores this P-VASP. Incubation of aortic tissue from New Zealand White Rabbits with the NOS inhibitor
N
G
-nitro-
l
d-arginine and endothelial removal strikingly reduced P-VASP. Oxidative stress induced by inhibition of CuZn superoxide dismutase increased superoxide and decreased P-VASP. Endothelial dysfunction in hyperlipidemic Watanabe rabbits (WHHL) was associated with increased vascular superoxide and with decreased P-VASP. Treatment of WHHL with AT
1
receptor blockade improved endothelial dysfunction, reduced vascular superoxide, increased vascular NO bioavailability, and increased P-VASP. Therefore, the level of vessel P-VASP closely follows changes in endothelial function and vascular oxidative stress. P-VASP is suggested to represent a novel biochemical marker for monitoring the NO-stimulated sGC/cGK-I pathway and endothelial integrity in vascular tissue.
Role of Endothelium-Derived Nitric Oxide in the Regulation of Cardiac Oxygen Metabolism
Authors: Trochu Jean-Noël , Bouhour Jean-Brieuc , Kaley Gabor , Hintze Thomas H.
Published: June 2012

Abstract
—Endothelium-derived NO is considered to be primarily an important determinant of vascular tone and platelet activity; however, the modulation of myocardial metabolism by NO may be one of its most important roles. This modulation may be critical for the regulation of tissue metabolism. Several physiological processes act in concert to make endothelial NO synthase–derived NO potentially important in the regulation of mitochondrial respiration in cardiac tissue, including (1) the nature of the capillary network in the myocardium, (2) the diffusion distance for NO, (3) the low toxicity of NO at physiological (nanomolar) concentrations, (4) the fact that low P
o
2
in tissue facilitates the action of NO on cytochrome oxidase, and (5) the formation of oxygen free radicals. A decrease in NO production is involved in the pathophysiological modifications that occur in heart failure and diabetes, disease states associated with altered cardiac metabolism that contributes to the evolution of the disease process. In contrast, several drugs (eg, angiotensin-converting enzyme inhibitors, amlodipine, and statins) can restore or maintain endogenous production of NO by endothelial cells, and this mechanism may explain part of their therapeutic efficiency. Thus, the purpose of this review is to critically evaluate the role of NO in the control of mitochondrial respiration, with special emphasis on its effect on cardiac metabolism.
A Role for Platelets and Endothelial Selectins in Tumor Necrosis Factor-α–Induced Leukocyte Recruitment in the Brain Microvasculature
Authors: Carvalho-Tavares Juliana , Hickey Michael J. , Hutchison Jamie , Michaud Jean , Sutcliffe Ian T. , Kubes Paul
Published: June 2012

Abstract
—The mechanisms mediating leukocyte recruitment into the cerebral nervous system during inflammation are still poorly understood. The objective of this study was to investigate the leukocyte recruitment in the brain microcirculation by intravital microscopy. Superfusion of the brain with artificial cerebrospinal fluid did not induce leukocyte rolling or adhesion. However, intraperitoneal tumor necrosis factor-α (TNF-α) caused marked leukocyte rolling and adhesion in the brain microcirculation. Histology revealed that the recruitment was primarily of neutrophils. Both E- and P-selectin were required for TNF-α–induced leukocyte recruitment, as rolling was reduced after treatment with either anti–E- or anti–P-selectin antibody and eliminated in E- or P-selectin–deficient mice. A significant increase in brain P- and E-selectin expression was seen after TNF-α treatment, but both were an order of magnitude less than in any other tissue. We observed significant platelet paving of TNF-α–stimulated endothelium and found that anti-platelet antibody reduced leukocyte rolling and adhesion, as did acetylsalicylic acid (aspirin). However, depletion of platelets did not reduce cerebral P-selectin expression. Moreover, chimeric mice lacking P-selectin on endothelium but not platelets had significantly decreased P-selectin expression and reduced leukocyte recruitment in the brain. This suggests a role for endothelial P-selectin in cerebral leukocyte recruitment. In conclusion, TNF-α–induced neutrophil recruitment into the brain requires both endothelial E-selectin and P-selectin as well as platelets, but platelet P-selectin was not a major contributor to this process.
Prolonged Hypercapnia-Evoked Cerebral Hyperemia via K
+
Channel– and Prostaglandin E
2
–Dependent Endothelial Nitric Oxide Synthase Induction
Authors: Najarian Taline , Marrache Anne Marilise , Dumont Isabelle , Hardy Pierre , Beauchamp Martin H. , Hou Xin , Peri Krishna , Gobeil Fernand , Varma Daya R. , Chemtob Sylvain
Published: June 2012

Abstract
—Mechanisms for secondary sustained increase in cerebral blood flow (CBF) during prolonged hypercapnia are unknown. We show that induction of endothelial NO synthase (eNOS) by an increase in prostaglandins (PGs) contributes to the secondary CBF increase during hypercapnic acidosis. Ventilation of pigs with 6% CO
2
(Pa
co
2≈
65 mm Hg; pH ≈7.2) caused a ≈2.5-fold increase in CBF at 30 minutes, which declined to basal values at 3 hours and gradually rose again at 6 and 8 hours; the latter increase was associated with PG elevation, nitrite formation, eNOS mRNA expression, and in situ NO synthase (NOS) reactivity (NADPH-diaphorase staining). Subjecting free-floating brain sections to acidotic conditions increased eNOS expression, the time course of which was similar to that of CBF increase. Treatment of pigs with the cyclooxygenase inhibitor diclofenac or the NOS inhibitor
Nω
-nitro-
l
-arginine blunted the initial rise and prevented the secondary CBF increase during hypercapnic acidosis; neuronal NOS blockers 1-(2-trifluoromethylphenyl) imidazole and 3-bromo-7-nitroindazole were ineffective. Diclofenac abolished the hypercapnia-induced rise in cerebrovascular nitrite production, eNOS mRNA expression, and NADPH-diaphorase reactivity. Acidosis (pH ≈7.15, P
co
2≈
40 mm Hg; 6 hours) produced similar increases in prostaglandin E
2
(PGE
2
) and eNOS mRNA levels in isolated brain microvessels and in NADPH-diaphorase reactivity of brain microvasculature; these changes were prevented by diclofenac, by the receptor-operated Ca
2+
channel blocker SK&F96365, and by the K
ATP
channel blocker glybenclamide. Acidosis increased Ca
2+
transients in brain endothelial cells, which were blocked by glybenclamide and SK&F96365 but not by diclofenac. Increased PG-related eNOS mRNA and NO-dependent vasorelaxation to substance P was detected as well in rat brain exposed to 6 hours of hypercapnia. PGE
2
was the only major prostanoid that modulated brain eNOS expression during acidosis. Thus, in prolonged hypercapnic acidosis, the secondary CBF rise is closely associated with induction of eNOS expression; this seems to be mediated by PGE
2
generated by a K
ATP
and Ca
2+
channel–dependent process.
Inhibition by Protein Kinase C of the K
NDP
Subtype of Vascular Smooth Muscle ATP-Sensitive Potassium Channel
Authors: Cole William C. , Malcolm Todd , Walsh Michael P. , Light Peter E.
Published: June 2012

Abstract
—ATP-sensitive K
+
channels (K
ATP
) contribute to the regulation of tone in vascular smooth muscle cells. We determined the effects of protein kinase C (PKC) activation on the nucleoside diphosphate–activated (K
NDP
) subtype of vascular smooth muscle K
ATP
channel. Phorbol 12,13-dibutyrate (PdBu) and angiotensin II inhibited K
NDP
activity of C-A patches of rabbit portal vein (PV) myocytes, but an inactive phorbol ester was without effect, and pretreatment with PKC inhibitor prevented the actions of PdBu. Constitutively active PKC inhibited K
NDP
in I-O patches but was without effect in the presence of a specific peptide inhibitor of PKC. PdBu increased the duration of a long-lived interburst closed state but was without effect on burst duration or intraburst kinetics. PdBu treatment inhibited K
NDP
, but not a 70-pS K
ATP
channel of rat PV. The results indicate that the K
NDP
subtype of vascular smooth muscle K
ATP
channel is inhibited by activation of PKC. Control of K
NDP
activity by intracellular signaling cascades involving PKC may, therefore, contribute to control of tone and arterial diameter by vasoconstrictors. (Circ Res. 2000;87:112-117.)
Morphological and Molecular Characterization of Adult Cardiomyocyte Apoptosis During Hypoxia and Reoxygenation
Authors: Kang Peter M. , Haunstetter Armin , Aoki Hiroki , Usheva Anny , Izumo Seigo
Published: June 2012

Abstract
—Apoptosis has been implicated in ischemic heart disease, but its mechanism in cardiomyocytes has not been elucidated. In this study, we investigate the effects of hypoxia and reoxygenation in adult cardiomyocytes and the molecular mechanism involved in cardiomyocyte apoptosis. Morphologically, reoxygenation induced rounding up of the cells, appearance of membrane blebs that were filled with marginated mitochondria, and ultrastructural findings characteristic of apoptosis. Reoxygenation (18 hours of reoxygenation after 6 hours of hypoxia) and prolonged hypoxia (24 hours of hypoxia) resulted in a 59% and 51% decrease in cellular viability, respectively. During reoxygenation, cell death occurred predominantly via apoptosis associated with appearance of cytosolic cytochrome
c
and activation of caspase-3 and -9. However, nonapoptotic cell death predominated during prolonged hypoxia. Both caspase inhibition and Bcl-2 overexpression during reoxygenation significantly improved cellular viability through inhibition of apoptosis but had minimal effect on hypoxia-induced cell death. Bcl-2 overexpression blocked reoxygenation-induced cytochrome
c
release and activation of caspase -3 and -9, but caspase inhibition alone did not block cytochrome
c
release. These results suggest that apoptosis predominates in cardiomyocytes after reoxygenation through a mitochondrion-dependent apoptotic pathway, and Bcl-2 prevents reoxygenation-induced apoptosis by inhibiting cytochrome
c
release from the mitochondria and prevents activation of caspase-3 and -9. (Circ Res. 2000;87:118-125.)
Release of Active Tissue Factor by Human Arterial Smooth Muscle Cells
Authors: Schecter Alison D. , Spirn Benjamin , Rossikhina Maria , Giesen Peter L. A. , Bogdanov Vladimir , Fallon John T. , Fisher Edward A. , Schnapp Lynn M. , Nemerson Yale , Taubman Mark B.
Published: June 2012

Abstract
—Tissue factor (TF), the initiator of coagulation, is thought to function predominantly at the cell surface. Recent data have suggested that active TF is present extracellularly in atherosclerotic plaques, the arterial wall, and the blood. This study was conducted to determine whether smooth muscle cells (SMCs), a major source of arterial TF, could generate extracellular TF. Active TF accumulated in the medium of cultured human SMCs, representing ≈10% of that measured in the underlying cells at 24 hours. Platelet-derived growth factor, phorbol ester, and tumor necrosis factor-α caused ≈3-fold increases in TF activity in the medium. Release of TF into the medium was dependent on the presence of the TF transmembrane domain but not the cytoplasmic domain. Antibodies to TF precipitated most of the activity from the culture medium, whereas antibodies to the β
1
-integrin subunit precipitated ≈33% of the activity. Treatment with detergent or phosphatidylserine:phosphatidylcholine did not increase activity, suggesting that all TF released by SMCs was in the appropriate lipid milieu and not encrypted. Western blotting showed that the medium contained full-length TF protein. Fluorescent cytometry showed that extracellular TF was present largely in particles ≤200 nm, which had a density of 1.10 g/mL. We hypothesize that active extracellular TF found in the injured arterial wall and atherosclerotic plaques derives, in part, from SMC microparticles. (Circ Res. 2000;87:126-132.)
Nitric Oxide Inhalation Decreases Pulmonary Artery Remodeling in the Injured Lungs of Rat Pups
Authors: Roberts Jesse D. , Chiche Jean-Daniel , Weimann Joerg , Steudel Wolfgang , Zapol Warren M. , Bloch Kenneth D.
Published: June 2012

Abstract
—Vascular injury causes the muscularization of peripheral pulmonary arteries, which is more pronounced in the infant than in the adult lung. Although inhaled NO gas attenuates pulmonary artery remodeling in hypoxic rats, whether or not it protects the lung by mitigating vasoconstriction is unknown. This investigation tested whether inhaled NO decreases the muscularization of injured pulmonary arteries in rat pups by modulating vascular tone. One week after monocrotaline administration, the percentage of muscularized rat pup lung arteries was increased by >3-fold. Nevertheless, monocrotaline exposure did not cause right ventricular hypertrophy, pulmonary hypertension, or vasoconstriction. In addition, it did not increase the expression of markers of inflammation (interleukin-1β, intercellular adhesion molecule-1, and E-selectin) or of platelet-mediated thrombosis (GPIbα). Continuous inhalation of 20 ppm NO gas prevented the neomuscularization of the pulmonary arteries in pups with lung injury. Moreover, a 3-fold increase in cell proliferation and 30% decrease in cell numbers in pulmonary arteries caused by monocrotaline exposure was prevented by NO inhalation. These data indicate that inhaled NO protects infants against pulmonary remodeling induced by lung injury by mechanisms that are independent of pulmonary tone, inflammation, or thrombosis. (Circ Res. 2000;87:140-145.)
Role of Vascular Cell Adhesion Molecule-1 and Fibronectin Connecting Segment-1 in Monocyte Rolling and Adhesion on Early Atherosclerotic Lesions
Authors: Huo Yuqing , Hafezi-Moghadam Ali , Ley Klaus
Published: June 2012

Abstract
—Atherosclerotic lesion development seems to be inflammatory in nature and involves the recruitment of monocytes to the vessel wall. In this study, we investigated the role of vascular cell adhesion molecule-1 (VCAM-1) and fibronectin (FN) connecting segment-1 containing the amino acid sequence ILDV as functional ligands for α
4
β
1
integrin (VLA-4) in monocyte rolling and adherence to early atherosclerotic lesions. Carotid arteries of apolipoprotein E–deficient mice were isolated and perfused with monocytes or U937 cells. Cell adhesion was reduced 95±4% by monoclonal antibodies HP1/2 and HP2/1, which block VLA-4 binding to both VCAM-1 and FN connecting segment-1. mAb HP1/3 preferentially blocked interaction of VLA-4 with FN but not VCAM-1 and decreased adhesion by 30±8%. In contrast, blocking VCAM-1 by perfusing the isolated carotid artery with mAb MK-2.7 reduced adhesion by 75±12%. Mononuclear cell adhesion to the early atherosclerotic endothelium was inhibited by 68±10% in the presence of EILDVPST but not in the presence of control peptide EIDVLPST. When VLA-4 or VCAM-1 was blocked, more mononuclear cells rolled on early lesions at significantly higher (approximately doubled) rolling velocities. These data demonstrate that (1) blockade of VCAM-1 can abrogate the majority (75±12%) of VLA-4–dependent monocyte adhesion on early atherosclerotic endothelia and (2) ILDV peptide interferes with VLA-4 binding to both VCAM-1 and FN and may be useful in limiting monocyte adhesion to atherosclerotic lesions. (Circ Res. 2000;87:153-159.)