bluuebunny/crossref_metadata_2025 huggingface dataset:
Ischemic Preconditioning Activates Phosphatidylinositol-3-Kinase Upstream of Protein Kinase C
Authors: Tong Haiyan , Chen Weina , Steenbergen Charles , Murphy Elizabeth
Published: June 2012

Abstract
—The present study is designed to test whether phosphatidylinositol 3-kinase (PI3-kinase) has a role in the signaling pathway in ischemic preconditioning (PC) and whether it is proximal or distal to protein kinase C (PKC). Before 20 minutes of global ischemia, Langendorff-perfused rat hearts were perfused for 20 minutes (control); preconditioned with 4 cycles of 5-minute ischemia and 5-minute reflow (PC); treated with either wortmannin (WM) or LY 294002 (LY), each of which is a PI3-kinase inhibitor, for 5 minutes before and throughout PC; treated with 1,2-dioctanoyl-
sn
-glycerol (DOG), an activator of PKC for 10 minutes (DOG); treated identically to the DOG group except with WM added 10 minutes before and during perfusion with DOG; or treated with either WM or LY for 25 minutes. Recovery of left ventricular developed pressure (LVDP; percentage of initial preischemic LVDP), measured after 30 minutes of reflow, was improved by PC (72±2% versus 36±4% in control;
P
<0.001), and this was blocked by WM and LY (41±4% and 43±5%, respectively;
P
<0.05 compared with PC). DOG addition improved postischemic LVDP (67±6%;
P
<0.001 compared with control), but in contrast to its effect on PC, WM did not completely eliminate the protective effect of DOG (52±4%;
P
>0.05 compared with DOG;
P
<0.05 compared with control). PC induced phosphorylation of protein kinase B and translocation of PKCε, and it increased NO production, and these effects were blocked by WM, which suggests a role for PI3-kinase in PC upstream of PKC and NO.
Human Brain Capillary Endothelium
Authors: Chen Ye , McCarron Richard M. , Ohara Yukoh , Bembry Joliet , Azzam Nabil , Lenz Fred A. , Shohami Esther , Mechoulam Raphael , Spatz Maria
Published: June 2012

Abstract
—In brain, the regulatory mechanism of the endothelial reactivity to nitric oxide and endothelin-1 may involve Ca
2+
, cytoskeleton, and vasodilator-stimulated phosphoprotein changes mediated by the cGMP/cGMP kinase system.

1

Endothelium of human brain capillaries or microvessels is used to examine the interplay of endothelin-1 with the putative vasorelaxant 2-arachidonoyl glycerol, an endogenous cannabimimetic derivative of arachidonic acid. This study demonstrates that 2-arachidonoyl glycerol counteracts Ca
2+
mobilization and cytoskeleton rearrangement induced by endothelin-1. This event is independent of nitric oxide, cyclooxygenase, and lipoxygenase and is mediated in part by cannabimimetic CB1 receptor, G protein, phosphoinositol signal transduction pathway, and Ca
2+
-activated K
+
channels. The induced rearrangements of cellular cytoskeleton (actin or vimentin) are partly prevented by inhibition of protein kinase C or high levels of potassium chloride. The 2-arachidonoyl glycerol–induced phosphorylation of vasodilator-stimulated phosphoprotein is mediated by cAMP. These findings suggest that 2-arachidonoyl glycerol may contribute to the regulation of cerebral capillary and microvascular function.
Activation of RhoA by Thrombin in Endothelial Hyperpermeability
Authors: Amerongen Geerten P. van Nieuw , Delft Sanne van , Vermeer Mario A. , Collard John G. , van Hinsbergh Victor W. M.
Published: June 2012

Abstract
—Endothelial cells (ECs) actively regulate the extravasation of blood constituents. On stimulation by vasoactive agents and thrombin, ECs change their cytoskeletal architecture and small gaps are formed between neighboring cells. These changes partly depend on a rise in [Ca
2+
]
i
and activation of the Ca
2+
/calmodulin-dependent myosin light chain kinase. In this study, mechanisms that contribute to the thrombin-enhanced endothelial permeability were further investigated. We provide direct evidence that thrombin induces a rapid and transient activation of RhoA in human umbilical vein ECs. Under the same conditions, the activity of the related protein Rac was not affected. This was accompanied by an increase in myosin light chain phosphorylation, the generation of F-actin stress fibers, and a prolonged increase in endothelial permeability. Inhibition of the RhoA target Rho kinase with the specific inhibitor Y-27632 reduced all of these effects markedly. In the presence of Y-27632, the thrombin-enhanced permeability was additionally reduced by chelation of [Ca
2+
]
i
by BAPTA. These data indicate that RhoA/Rho kinase and Ca
2+
represent 2 pathways that act on endothelial permeability. In addition, the protein tyrosine kinase inhibitor genistein reduced thrombin-induced endothelial permeability without affecting activation of RhoA by thrombin. Our data support a model of thrombin-induced endothelial permeability that is regulated by 3 cellular signal transduction pathways.
Potential Antiatherogenic Mechanisms of Ascorbate (Vitamin C) and α-Tocopherol (Vitamin E)
Authors: Carr Anitra C. , Zhu Ben-Zhan , Frei Balz
Published: June 2012

Abstract
—The premise that oxidative stress, among several other factors, plays an important role in atherogenesis implies that the development and progression of atherosclerosis can be inhibited by antioxidants. In this minireview we discuss several mechanisms by which the antioxidants ascorbate (vitamin C) and α-tocopherol (vitamin E) may protect against atherosclerosis. These mechanisms include inhibition of LDL oxidation and inhibition of leukocyte adhesion to the endothelium and vascular endothelial dysfunction. Overall, ascorbate appears to be more effective than α-tocopherol in mitigating these pathophysiological processes, most likely as a result of its abilities to effectively scavenge a wide range of reactive oxygen and nitrogen species and to regenerate α-tocopherol, and possibly tetrahydrobiopterin, from its radical species. In contrast, α-tocopherol can act either as an antioxidant or a pro-oxidant to inhibit or facilitate, respectively, lipid peroxidation in LDL. However, this pro-oxidant activity of α-tocopherol is prevented by ascorbate acting as a coantioxidant. Therefore, an optimum vitamin C intake or body status may help protect against atherosclerosis and its clinical sequelae, whereas vitamin E may only be effective in combination with vitamin C.
Increased Nitric Oxide Synthase Activity and Expression in the Human Uterine Artery During Pregnancy
Authors: Nelson Sharon H. , Steinsland Odd S. , Wang Yu , Yallampalli Chandra , Dong Y.-L. , Sanchez Jennifer M.
Published: June 2012

Abstract
—Evidence exists that NO plays a role in the vasodilation that occurs during pregnancy. The purpose of the present study was to determine whether the role of NO is associated with an increase in the activity and protein expression of NO synthase (NOS) in the human uterine artery. Uterine arteries were obtained from pregnant patients (P arteries) and nonpregnant patients (NP arteries). NOS activity was estimated with the
l
-[
3
H]-arginine–to–
l
-[
3
H]-citrulline conversion method and on the basis of changes in tissue levels of cGMP. Western immunoblotting and immunohistochemistry were used to assess NOS protein expression. Ca
2+
-dependent NOS activity was 8 times greater (
P
<0.01) in P than in NP arteries. Although most of this pregnancy-induced increase in NOS activity was Ca
2+
dependent (64%), a considerable portion was Ca
2+
independent. Expressions of endothelial NOS (eNOS) and neuronal NOS, but not inducible NOS, were demonstrated in P and NP arteries. The eNOS was located in the endothelium and stained with a qualitative order of P arteries>NP arteries (follicular)>NP arteries (luteal). The neuronal NOS was located in the adventitia of P and NP arteries. Basal NO-dependent and bradykinin-stimulated levels of cGMP were higher (
P
<0.05) in P than in NP arteries. These results indicate that an upregulation of eNOS protein expression could account for the increased NO synthesis/release in the human uterine artery during pregnancy.
A Novel Angiotensin-Converting Enzyme–Related Carboxypeptidase (ACE2) Converts Angiotensin I to Angiotensin 1-9
Authors: Donoghue Mary , Hsieh Frank , Baronas Elizabeth , Godbout Kevin , Gosselin Michael , Stagliano Nancy , Donovan Michael , Woolf Betty , Robison Keith , Jeyaseelan Raju , Breitbart Roger E. , Acton Susan
Published: June 2012

Abstract
—ACE2, the first known human homologue of angiotensin-converting enzyme (ACE), was identified from 5′ sequencing of a human heart failure ventricle cDNA library. ACE2 has an apparent signal peptide, a single metalloprotease active site, and a transmembrane domain. The metalloprotease catalytic domains of ACE2 and ACE are 42% identical, and comparison of the genomic structures indicates that the two genes arose through duplication. In contrast to the more ubiquitous ACE, ACE2 transcripts are found only in heart, kidney, and testis of 23 human tissues examined. Immunohistochemistry shows ACE2 protein predominantly in the endothelium of coronary and intrarenal vessels and in renal tubular epithelium. Active ACE2 enzyme is secreted from transfected cells by cleavage N-terminal to the transmembrane domain. Recombinant ACE2 hydrolyzes the carboxy terminal leucine from angiotensin I to generate angiotensin 1-9, which is converted to smaller angiotensin peptides by ACE in vitro and by cardiomyocytes in culture. ACE2 can also cleave des-Arg bradykinin and neurotensin but not bradykinin or 15 other vasoactive and hormonal peptides tested. ACE2 is not inhibited by lisinopril or captopril. The organ- and cell-specific expression of ACE2 and its unique cleavage of key vasoactive peptides suggest an essential role for ACE2 in the local renin-angiotensin system of the heart and kidney. The full text of this article is available at http://www.circresaha.org.
Endothelial Cell Apoptosis in Angiogenesis and Vessel Regression
Authors: Dimmeler Stefanie , Zeiher Andreas M.
Published: June 2012

Abstract
—The programmed form of cell death (apoptosis) is essential for normal development of multicellular organisms. In the past few years, compelling evidence accumulated that dysregulation of apoptosis can lead to embryonal death and is involved in the pathophysiology of various inflammatory and degenerative diseases. Specifically, the occurrence of endothelial cell apoptosis has deleterious effects on the development of the cardiovascular system leading to embryonal death. Moreover, endothelial cell apoptosis counteracts neovascularization in the adult organism. On the basis of these findings, one may consider the regulation of endothelial cell apoptosis as a potential therapeutic target. The induction of endothelial cell apoptosis may limit unwanted neovascularization of tumors. In contrast, the prevention of endothelial cell apoptosis may improve angiogenesis and vasculogenesis in patients with ischemia. The present work critically reviews the existing data that supports a role of endothelial cell apoptosis for vascular growth and remodeling and provides insights into the mechanisms and the potential therapeutic consequences.
Opening of Mitochondrial K
ATP
Channels Triggers the Preconditioned State by Generating Free Radicals
Authors: Pain Tilley , Yang Xi-Ming , Critz Stuart D. , Yue Yankun , Nakano Atsushi , Liu Guang S. , Heusch Gerd , Cohen Michael V. , Downey James M.
Published: June 2012

Abstract
—The critical time for opening mitochondrial (mito) K
ATP
channels, putative end effectors of ischemic preconditioning (PC), was examined. In isolated rabbit hearts 29±3% of risk zone infarcted after 30 minutes of regional ischemia. Ischemic PC or 5-minute exposure to 10 μmol/L diazoxide, a mito K
ATP
channel opener, reduced infarction to 3±1% and 8±1%, respectively. The mito K
ATP
channel closer 5-hydroxydecanoate (200 μmol/L), bracketing either 5-minute PC ischemia or diazoxide infusion, blocked protection (24±3 and 28±6% infarction, respectively). However, 5-hydroxydecanoate starting 5 minutes before long ischemia did not affect protection. Glibenclamide (5 μmol/L), another K
ATP
channel closer, blocked the protection by PC only when administered early. These data suggest that K
ATP
channel opening triggers protection but is not the final step. Five minutes of diazoxide followed by a 30-minute washout still reduced infarct size (8±3%), implying memory as seen with other PC triggers. The protection by diazoxide was not blocked by 5 μmol/L chelerythrine, a protein kinase C antagonist, given either to bracket diazoxide infusion or just before the index ischemia. Bracketing preischemic exposure to diazoxide with 50 μmol/L genistein, a tyrosine kinase antagonist, did not affect infarction, but genistein blocked the protection by diazoxide when administered shortly before the index ischemia. Thus, although it is not protein kinase C-dependent, the protection by diazoxide involves tyrosine kinase. Bracketing diazoxide perfusion with
N
-(2-mercaptopropionyl) glycine (300 μmol/L) or Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (7 μmol/L), each of which is a free radical scavenger, blocked protection, indicating that diazoxide triggers protection through free radicals. Therefore, mito K
ATP
channels are not the end effectors of protection, but rather their opening before ischemia generates free radicals that trigger entrance into a preconditioned state and activation of kinases.
Expression and Function of Endothelial Ca
2+
-Activated K
+
Channels in Human Mesenteric Artery
Authors: Köhler Ralf , Degenhardt Christiane , Kühn Meike , Runkel Norbert , Paul Martin , Hoyer Joachim
Published: June 2012

Abstract
—Ca
2+
-activated K
+
(K
Ca
) channels have been suggested to play a role in the control of endothelial functions such as regulation of vascular tone and cell proliferation. We established a method for single-cell reverse transcriptase–polymerase chain reaction analysis in combination with the patch-clamp technique to characterize K
Ca
channel expression and function in single endothelial cells (ECs) within the endothelial monolayer of intact human mesenteric arteries (MAs) and in disease states. We tested whether endothelial K
Ca
channel expression and function are altered in MAs obtained from patients with colonic adenocarcinoma (CA) compared with those in MAs from non–cancer patients with inactive diverticulitis. Expression of the intermediate-conductance K
Ca
channel (hIK1) was detected in non–cancer and CA patients. In whole-cell patch-clamp measurements, only ECs expressing hIK1 exhibited corresponding K
Ca
currents, whereas respective K
Ca
currents were missing in hIK1-negative ECs. This heterogeneity of hIK1 expression patterns is indicative of a specialized subset of ECs within the endothelial monolayer. In CA patients, compared with non–cancer patients, a 2.5-fold increase in hIK1-expressing ECs per MA was observed (
P
<0.05). However, K
Ca
current densities in hIK1-expressing ECs of both groups were similar. In addition to hIK1, expression of the large-conductance K
Ca
channel (hSlo) was detected in single ECs from CA patients. The increased K
Ca
channel expression in CA patients resulted in a 2.7-fold increase of bradykinin-induced endothelial hyperpolarization compared with controls (
P
<0.05). This increased expression and function of K
Ca
channels might indicate an altered functional state of the endothelium in cancer patients and could play a role in tumor angiogenesis.
The Preconditioning Phenomenon
Authors: Yellon Derek M. , Dana Ali
Published: June 2012

Abstract
—The possibility that an innate mechanism of myocardial protection might be inducible in the human heart has generated considerable excitement and enthusiastic research. The potential to enhance myocardial resistance to ischemic injury in patients suffering the consequences of coronary artery disease has led to studies with more direct clinical relevance. However, in common with many other areas of clinical interest based on advances in basic scientific understanding, early enthusiasm may be disproportionate to ultimate therapeutic significance. There can be little doubt that our understanding of the mechanisms underlying the pathogenesis of ischemia-reperfusion injury has been enhanced significantly by the plethora of research stimulated by interest in endogenous myocardial protection. Direct extrapolation of observations in the laboratory to the cardiology clinic or operating theater is tempting but should be avoided. The results of recent clinical experiments that suggest that preconditioning can protect against ischemia, although encouraging, should be interpreted cautiously, with particular attention to the limitations of the end points available. A reasoned evaluation of recent research should prevent unrealistic expectations and allow improved design of future trials so that this potent adaptive phenomenon can be exploited to its maximum potential.
Oral Administration of Tetrahydrobiopterin Prevents Endothelial Dysfunction and Vascular Oxidative Stress in the Aortas of Insulin-Resistant Rats
Authors: Shinozaki Kazuya , Nishio Yoshihiko , Okamura Tomio , Yoshida Yuichi , Maegawa Hiroshi , Kojima Hideto , Masada Masahiro , Toda Noboru , Kikkawa Ryuichi , Kashiwagi Atsunori
Published: June 2012

Abstract
—We have reported that a deficiency of tetrahydrobiopterin (BH
4
), an active cofactor of endothelial NO synthase (eNOS), contributes to the endothelial dysfunction through reduced eNOS activity and increased superoxide anion (O
2
−
) generation in the insulin-resistant state. To further confirm this hypothesis, we investigated the effects of dietary treatment with BH
4
on endothelium-dependent arterial relaxation and vascular oxidative stress in the aortas of insulin-resistant rats. Oral supplementation of BH
4
(10 mg · kg
−1
· d
−1
) for 8 weeks significantly increased the BH
4
content in cardiovascular tissues of rats fed high levels of fructose (fructose-fed rats). Impairment of endothelium-dependent arterial relaxation in the aortic strips of the fructose-fed rats was reversed with BH
4
treatment. The BH
4
treatment was associated with a 2-fold increase in eNOS activity as well as a 70% reduction in endothelial O
2
−
production compared with those in fructose-fed rats. The BH
4
treatment also partially improved the insulin sensitivity and blood pressure, as well as the serum triglyceride concentration, in the fructose-fed rats. Moreover, BH
4
treatment of the fructose-fed rats markedly reduced the lipid peroxide content of both aortic and cardiac tissues and inhibited the activation of 2 redox-sensitive transcription factors, nuclear factor-κB and activating protein-1, which were increased in fructose-fed rats. The BH
4
treatment of control rats did not have any significant effects on these parameters. These results indicate that BH
4
augmentation is essential for the restoration of eNOS function and the reduction of vascular oxidative stress in insulin-resistant rats.
Hyperglycemia Inhibits Vascular Smooth Muscle Cell Apoptosis Through a Protein Kinase C–Dependent Pathway
Authors: Hall Jennifer L. , Matter Christian M. , Wang Xiaohong , Gibbons Gary H.
Published: June 2012

Abstract
—We hypothesized that the pathogenesis of diabetic vasculopathy involves the abnormal regulation of vascular smooth muscle cell (VSMC) apoptosis. In nondiabetic mice, a reduction in carotid artery blood flow resulted in a significant loss of medial VSMCs via apoptosis (normal flow 84±1 viable VSMCs, reduced flow 70±5 viable VSMCs; n=12,
P
<0.01). In contrast, flow-induced VSMC apoptosis was markedly attenuated in streptozotocin-induced diabetic mice (normal flow 85±2 viable VSMC, reduced flow 82±4 viable VSMC; n=13, NS). In accord with our in vivo findings, the exposure of cultured rat and human VSMCs to high glucose (17.5 mmol/L) significantly attenuated the induction of apoptosis in response to serum withdrawal (rat VSMCs in normal [5.5 mmol/L] glucose 28±1%, high
d
-glucose 19±2%;
P
<0.0001). High glucose also inhibited apoptosis induced by Fas ligand (100 ng/mL) (normal 23±2%, high
d
-glucose 13±2%;
P
<0.006). Supplementation with the nonmetabolized enantiomer
l
-glucose had no effect. We confirmed reports that high glucose activates protein kinase C (PKC) and demonstrated that PKC blockade with long-term phorbol ester treatment or calphostin C prevented the antiapoptotic effect (
P
<0.001). Moreover, the upregulation of either PKCα or PKCβII expression was sufficient to inhibit serum withdrawal–induced apoptosis (control 25±2%, PKCα 11±2%, PKCβII 8±2%;
P
<0.0001), whereas the upregulation of PKCδ had no significant effect. Taken together, these findings demonstrate that hyperglycemia inhibits VSMC apoptosis via a PKC-dependent pathway.
Angiopoietin-1 Is an Antipermeability and Anti-Inflammatory Agent In Vitro and Targets Cell Junctions
Authors: Gamble Jennifer R. , Drew Jenny , Trezise Libby , Underwood Anne , Parsons Michelle , Kasminkas Lisa , Rudge John , Yancopoulos George , Vadas Mathew A.
Published: June 2012

Abstract
—Inflammation is a basic pathological mechanism that underlies many diseases. An important component of the inflammatory response is the passage of plasma components and leukocytes from the blood vessel into the tissues. The endothelial monolayer lining blood vessels reacts to stimuli such as thrombin or vascular endothelial growth factor by changes in cell-cell junctions, an increase in permeability, and the leakage of plasma components into tissues. Other stimuli, such as tumor necrosis factor-α (TNF-α), are responsible for stimulating the transmigration of leukocytes. Here we show that angiopoietin-1, a cytokine essential in fetal angiogenesis, not only supports the localization of proteins such as platelet endothelial cell adhesion molecule-1 (PECAM-1) into junctions between endothelial cells and decreases the phosphorylation of PECAM-1 and vascular endothelial cadherin, but it also strengthens these junctions, as evidenced by a decrease in basal permeability and inhibition of permeability responses to thrombin and vascular endothelial growth factor. Furthermore, angiopoietin-1 inhibits TNF-α–stimulated leukocyte transmigration. Angiopoietin-1 may thus have a major role in maintaining the integrity of endothelial monolayers.
Phosphate Regulation of Vascular Smooth Muscle Cell Calcification
Authors: Jono Shuichi , McKee Marc D. , Murry Charles E. , Shioi Atsushi , Nishizawa Yoshiki , Mori Katsuhito , Morii Hirotoshi , Giachelli Cecilia M.
Published: June 2012

Abstract
—Vascular calcification is a common finding in atherosclerosis and a serious problem in diabetic and uremic patients. Because of the correlation of hyperphosphatemia and vascular calcification, the ability of extracellular inorganic phosphate levels to regulate human aortic smooth muscle cell (HSMC) culture mineralization in vitro was examined. HSMCs cultured in media containing normal physiological levels of inorganic phosphate (1.4 mmol/L) did not mineralize. In contrast, HSMCs cultured in media containing phosphate levels comparable to those seen in hyperphosphatemic individuals (>1.4 mmol/L) showed dose-dependent increases in mineral deposition. Mechanistic studies revealed that elevated phosphate treatment of HSMCs also enhanced the expression of the osteoblastic differentiation markers osteocalcin and Cbfa-1. The effects of elevated phosphate on HSMCs were mediated by a sodium-dependent phosphate cotransporter (NPC), as indicated by the ability of the specific NPC inhibitor phosphonoformic acid, to dose dependently inhibit phosphate-induced calcium deposition as well as osteocalcin and Cbfa-1 gene expression. With the use of polymerase chain reaction and Northern blot analyses, the NPC in HSMCs was identified as Pit-1 (Glvr-1), a member of the novel type III NPCs. These data suggest that elevated phosphate may directly stimulate HSMCs to undergo phenotypic changes that predispose to calcification and offer a novel explanation of the phenomenon of vascular calcification under hyperphosphatemic conditions. The full text of this article is available at http://www.circresaha.org.
Laminar Shear Stress Upregulates Integrin Expression
Authors: Urbich Carmen , Walter Dirk H. , Zeiher Andreas M. , Dimmeler Stefanie
Published: June 2012

Abstract
—Laminar shear stress exerts important effects on endothelial cell (EC) function and inhibits apoptosis of ECs induced by various stimuli. The mechanism by which hemodynamic forces, such as shear stress, are transduced into cellular signaling is still not known. Located at the cell surface, integrins, which are required for cell adhesion and cell survival, are potential mechanotransducers. Therefore, we investigated the effect of shear stress on integrin expression in ECs. Shear stress time-dependently increased the mRNA expression of the fibronectin receptor subunits α
5
and β
1
with a maximum at 6 hours (283±41% and 215±27% of control, respectively). In addition, the protein levels of the fibronectin receptor subunits α
5
and β
1
were enhanced with a maximum at 12 hours of shear stress exposure (343±53% and 212±38% of control, respectively). The shear stress–induced upregulation of integrins is independent of nitric oxide. Furthermore, we confirmed the enhanced functional activity of α
5
β
1
integrin expression by FACS analysis. As a functional consequence, human umbilical vein ECs, which were preexposed to shear stress, revealed a significantly increased attachment (178±10% of static controls) and a more pronounced extracellular signal–regulated kinase 1 and 2 activation in response to cell attachment. Finally, we demonstrated that shear stress requires RGD-sensitive integrins to mediate its antiapoptotic effect. Taken together, these results define a novel mechanism by which shear stress may exert its atheroprotective effects via upregulation of integrins to support EC adhesion and survival.
Calcineurin and Beyond
Authors: Molkentin Jeffery D.
Published: June 2012

Abstract
—In response to increased ventricular wall tension or neurohumoral stimuli, the myocardium undergoes an adaptive hypertrophy response that temporarily augments pump function. Although initially beneficial, sustained cardiac hypertrophy can lead to decompensation and cardiomyopathy. Recent studies have focused on characterizing the molecular mechanisms that underlie cardiac hypertrophy. An increasing number of signal transduction pathways have been identified as important regulators of the hypertrophic response, including the low–molecular weight GTPases (Ras, RhoA, and Rac), mitogen-activated protein kinases, protein kinase C, and calcineurin. This review will discuss an emerging body of evidence that implicates the calcium-calmodulin–activated protein phosphatase calcineurin as a physiological regulator of the cardiac hypertrophic response. Although the sufficiency of calcineurin to promote cardiomyocyte hypertrophy in vivo and in vitro is established, its overall necessity as a hypertrophic mediator is currently an area of ongoing debate. The use of the calcineurin-inhibitory agents cyclosporine A and FK506 have suggested a necessary role for calcineurin in many, but not all, animal models of hypertrophy or cardiomyopathy. The evidence implicating a role for calcineurin signaling in the heart will be weighed against a growing body of literature suggesting necessary roles for a diverse array of intracellular signaling pathways, highlighting the multifactorial nature of the hypertrophic program.
Sinoatrial Node Pacemaker Activity Requires Ca
2+
/Calmodulin-Dependent Protein Kinase II Activation
Authors: Vinogradova Tatiana M. , Zhou Ying-Ying , Bogdanov Konstantin Y. , Yang Dongmei , Kuschel Meike , Cheng Heping , Xiao Rui-Ping
Published: June 2012

Abstract
—Cardiac beating arises from the spontaneous rhythmic excitation of sinoatrial (SA) node cells. Here we report that SA node pacemaker activity is critically dependent on Ca
2+
/calmodulin-dependent protein kinase II (CaMKII). In freshly dissociated rabbit single SA node cells, inhibition of CaMKII by a specific peptide inhibitor, autocamtide-2 inhibitory peptide (AIP, 10 μmol/L), or by KN-93 (0.1 to 3.0 μmol/L), but not its inactive analog, KN-92, depressed the rate and amplitude of spontaneous action potentials (APs) in a dose-dependent manner. Strikingly, 10 μmol/L AIP and 3 μmol/L KN-93 completely arrested SA node cells, which indicates that basal CaMKII activation is obligatory to the genesis of pacemaker AP. To understand the ionic mechanisms of the CaMKII effects, we measured L-type Ca
2+
current (
I
Ca, L
), which contributes both to AP upstroke and to pacemaker depolarization. KN-93 (1 μmol/L), but not its inactive analog, KN-92, decreased
I
Ca, L
amplitude from 12±2 to 6±1 pA/pF without altering the shape of the current-voltage relationship. Both AIP and KN-93 shifted the midpoint of the steady-state inactivation curve leftward and markedly slowed the recovery of
I
Ca, L
from inactivation. Similar results were observed using the fast Ca
2+
chelator BAPTA, whereas the slow Ca
2+
chelator EGTA had no significant effect, which suggests that CaMKII activity is preferentially regulated by local Ca
2+
transients. Indeed, confocal immunocytochemical imaging showed that active CaMKII is highly localized beneath the surface membrane in the vicinity of L-type channels and that AIP and KN-93 significantly reduced CaMKII activity. Thus, we conclude that CaMKII plays a vital role in regulating cardiac pacemaker activity mainly via modulating
I
Ca, L
inactivation and reactivation, and local Ca
2+
is critically involved in these processes.
Transgenic Modeling of a Cardiac Troponin I Mutation Linked to Familial Hypertrophic Cardiomyopathy
Authors: James Jeanne , Zhang Yan , Osinska Hanna , Sanbe Atsushi , Klevitsky Raisa , Hewett Timothy E. , Robbins Jeffrey
Published: June 2012

Abstract
—Multiple mutations in cardiac troponin I (cTnI) have been associated with familial hypertrophic cardiomyopathy. Two mutations are located in the cTnI inhibitory domain, a highly negatively charged region that alternately binds to either actin or troponin C, depending on the intracellular concentration of calcium. This region is critical to the inhibition of actin-myosin crossbridge formation when intracellular calcium is low. We modeled one of the inhibitory domain mutations, arginine145→glycine (TnI
146Gly
in the mouse sequence), by cardiac-specific expression of the mutated protein in transgenic mice. Multiple lines were generated with varying degrees of expression to establish a dose relationship; the severity of phenotype could be correlated directly with transgene expression levels. Transgenic mice overexpressing wild-type cTnI were generated as controls and analyzed in parallel with the TnI
146Gly
animals. The control mice showed no abnormalities, indicating that the phenotype of TnI
146Gly
was not simply an artifact of transgenesis. In contrast, TnI
146Gly
mice showed cardiomyocyte disarray and interstitial fibrosis and suffered premature death. The functional alterations that seem to be responsible for the development of cardiac disease include increased skinned fiber sensitivity to calcium and, at the whole organ level, hypercontractility with diastolic dysfunction. Severely affected lines develop a pathology similar to human familial hypertrophic cardiomyopathy but within a dramatically shortened time frame. These data establish the causality of this mutation for cardiac disease, provide an animal model for understanding the resultant pathogenic structure-function relationships, and highlight the differences in phenotype severity of the troponin mutations between human and mouse hearts.
Endothelium-Derived Hyperpolarizing Factor Synthase (Cytochrome P450 2C9) Is a Functionally Significant Source of Reactive Oxygen Species in Coronary Arteries
Authors: Fleming Ingrid , Michaelis U. Ruth , Bredenkötter Daniel , Fisslthaler Beate , Dehghani Faramarz , Brandes Ralf P. , Busse Rudi
Published: June 2012

Abstract
—In the porcine coronary artery, a cytochrome P450 (CYP) isozyme homologous to CYP 2C8/9 has been identified as an endothelium-derived hyperpolarizing factor (EDHF) synthase. As some CYP enzymes are reported to generate reactive oxygen species (ROS), we hypothesized that the coronary EDHF synthase may modulate vascular homeostasis by the simultaneous production of ROS and epoxyeicosatrienoic acids. In bradykinin-stimulated coronary arteries, antisense oligonucleotides against CYP 2C almost abolished EDHF-mediated responses but potentiated nitric oxide (NO)-mediated relaxation. The selective CYP 2C9 inhibitor sulfaphenazole and the superoxide anion (O
2
−
) scavengers Tiron and nordihydroguaretic acid also induced a leftward shift in the NO-mediated concentration-relaxation curve to bradykinin. CYP activity and O
2
−
production, determined in microsomes prepared from cells overexpressing CYP 2C9, were almost completely inhibited by sulfaphenazole. Sulfaphenazole did not alter the activity of either CYP 2C8, the leukocyte NADPH oxidase, or xanthine oxidase. ROS generation in coronary artery rings, visualized using either ethidium or dichlorofluorescein fluorescence, was detected under basal conditions. The endothelial signal was attenuated by CYP 2C antisense treatment as well as by sulfaphenazole. In isolated coronary endothelial cells, bradykinin elicited a sulfaphenazole-sensitive increase in ROS production. Although 11,12 epoxyeicosatrienoic acid attenuated the activity of nuclear factor-κB in cultured human endothelial cells, nuclear factor-κB activity was enhanced after the induction or overexpression of CYP 2C9, as was the expression of vascular cell adhesion molecule-1. These results suggest that a CYP isozyme homologous to CYP 2C9 is a physiologically relevant generator of ROS in coronary endothelial cells and modulates both vascular tone and homeostasis.
Functional Evidence for an Inward Rectifier Potassium Current in Rat Renal Afferent Arterioles
Authors: Chilton Lisa , Loutzenhiser Rodger
Published: June 2012

Abstract
—An inward rectifier potassium current, Kir, has been identified in cerebral and coronary resistance vessels, where it is considered to be an important determinant of resting membrane potential (RMP) and to play a role in blood flow regulation. We investigated the functional role of Kir in the renal afferent arteriole using the in vitro–perfused hydronephrotic rat kidney. Increasing external KCl from 5 to 15 mmol/L induced afferent arteriolar vasodilation. This response was inhibited by 10 to 100 μmol/L Ba
2+
, concentrations selective for blockade of Kir, and by chloroethylclonidine (100 μmol/L) but was not blocked by glibenclamide (10 μmol/L) or ouabain (3 mmol/L). Reducing external KCl from 5 to 1.5 mmol/L to enhance rectification of Kir caused vasoconstriction at low renal arterial pressure (40 mm Hg) and vasodilation during myogenic vasoconstriction (120 mm Hg), suggesting that this current dominates RMP at low perfusion pressures. When administered to kidneys perfused at 40 mm Hg renal arterial pressure, 30 μmol/L Ba
2+
elicited afferent arteriolar depolarization, reducing RMP from −47±2 to −34±2 mV (n=10,
P
<0.0001), and vasoconstriction, reducing diameters from 14.5±1 to 10.9±0.8 μm (n=10,
P
=0.0016). Although Ba
2+
reduced resting diameter, blockade of Kir did not prevent myogenic signaling in this vessel. Our findings thus demonstrate the presence of Kir in rat renal afferent arterioles and suggest that this current is an important determinant of RMP in situ.
Mouse Carotid Artery Ligation Induces Platelet-Leukocyte–Dependent Luminal Fibrin, Required for Neointima Development
Authors: Kawasaki Tomihisa , Dewerchin Mieke , Lijnen H. Roger , Vreys I. , Vermylen Jos , Hoylaerts Marc F.
Published: June 2012

Abstract
—The relationship between platelet and leukocyte activation, coagulation, and neointima development was investigated in noninjured murine blood vessels subjected to blood stasis. The left common carotid artery of C57BL/6J mice was ligated proximal to the bifurcation. Tissue-factor expression in luminal leukocytes progressively increased over 2 weeks. On day 3 after ligation, in addition to infiltrated granulocytes, platelet microthrombi and platelet-covered leukocytes as well as tissue-factor–positive fibrin deposits lined the endothelium. Maximal neointima formation in carotid artery cross sections of control mice equaled 28±3.7% (n=11) and 42±5.1% (n=8) of the internal elastic lamina cross-sectional area 1 and 2 weeks after ligation. In FVIII
−/−
mice, stenosis was significantly lower 1 (11±3.6%, n=8) and 2 (21±4.7%, n=7) weeks after ligation (both
P
<0.01 versus background-matched controls). In u-PA
−/−
mice, luminal stenosis was significantly higher 1 (38±7.0%, n=7) and 2 (77±5.6%, n=6) weeks after ligation (
P
<0.05 and
P
<0.01, respectively, versus matched controls). In α
2
-AP
−/−
mice, stenosis was lower at 1 week (14±2.6%, n=7,
P
<0.01) but not at 2 weeks. Responses in tissue-type plasminogen activator or plasminogen activator inhibitor-1 gene–deficient mice equaled that in controls. Reducing plasma fibrinogen levels in controls with ancrod or inducing partial thrombocytopenia with busulfan resulted in significantly less neointima, but inflammation was inhibited only in busulfan-treated mice. We conclude that stasis induces platelet activation, leading to microthrombosis and platelet-leukocyte conjugate formation, triggering inflammation and tissue-factor accumulation on the carotid artery endothelium. Delayed coagulation then results in formation of a fibrin matrix, which is used by smooth muscle cells to migrate into the lumen.
Role of Phasic Dynamism of p38 Mitogen-Activated Protein Kinase Activation in Ischemic Preconditioning of the Canine Heart
Authors: Sanada Shoji , Kitakaze Masafumi , Papst Philip J. , Hatanaka Kazuhito , Asanuma Hiroshi , Aki Toshihiko , Shinozaki Yoshiro , Ogita Hisakazu , Node Koichi , Takashima Seiji , Asakura Masanori , Yamada Junko , Fukushima Tomi , Ogai Akiko , Kuzuya Tsunehiko , Mori Hidezo , Terada Naohiro , Yoshida Ken-ichi , Hori Masatsugu
Published: June 2012

Abstract
—Although ischemic stress, including ischemic preconditioning (IP), activates p38 mitogen-activated protein kinase (MAPK), the relationship between p38 MAPK activation and the underlying cellular mechanisms of cardioprotection by IP is not verified in vivo. We examined the effects of the selective p38 MAPK inhibition on the cardioprotective effect of IP in the open-chest dogs. The coronary artery was occluded 4 times for 5 minutes, separated by 5 minutes of reperfusion (IP) followed by 90 minutes of occlusion and 6 hours of reperfusion. We infused SB203580 into the coronary artery during IP and 1 hour of reperfusion, during IP alone, and during sustained ischemia in the IP group. p38 MAPK activity markedly increased during IP but did not additionally increase at the onset of ischemia and was even attenuated at 15 minutes of sustained ischemia, and heat-shock protein (HSP) 27 was phosphorylated and translocated from cytosol to myofibril or nucleus without affecting total protein level at the onset of ischemia compared with the control group. SB203580 treatment (1 μmol/L) only during IP blunted the infarct size limitation by IP (37.3±6.3% versus 7.4±2.1% in the IP group,
P
<0.01) and attenuated either phosphorylation or translocation of HSP27 during IP. Although the SB203580 treatment throughout the preischemic and postischemic periods had no significant effect on infarct size (33.3±9.4%) in this model, treatment with SB203580 only during ischemia partially mimicked the infarct size limitation by IP (26.8±3.5%). Thus, transient p38 MAPK activation during ischemic preconditioning mainly mediates the cardioprotection followed by HSP27 phosphorylation and translocation in vivo in the canine heart.
Thrombogenic Effects of Antiphospholipid Antibodies Are Mediated by Intercellular Cell Adhesion Molecule-1, Vascular Cell Adhesion Molecule-1, and P-Selectin
Authors: Pierangeli Silvia S. , Espinola Ricardo G. , Liu Xiaowei , Harris E. Nigel
Published: June 2012

Abstract
—Recent studies have shown that antiphospholipid (aPL) enhances expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin on endothelial cells (ECs) and that these effects are correlated with increased adhesion of leukocytes to endothelium in cremaster muscle in vivo and with thrombosis in a mouse model. Activation of ECs by aPL may create a hypercoagulable state that precedes and contributes to thrombosis in patients with aPL syndrome (APS). This study proposed to examine whether this in vivo activation of ECs and enhanced thrombosis by aPL are mediated by ICAM-1, P-selectin, or VCAM-1. The dynamics of thrombus formation and the number of adhering leukocytes were studied in ICAM-1–deficient (ICAM-1
−/−
) mice or ICAM-1–/P-selectin–deficient (ICAM-1
−/−
/P-selectin
−/−
) mice treated with affinity-purified aPL antibodies (ap IgG-APS) or with control IgG and compared with wild-type mice treated in a similar fashion. In another set of experiments, the adhesion of leukocytes to cremaster muscle and the dynamics of thrombus formation were studied in CD1 mice treated with aPL or control IgG before and 30 minutes after intravenous infusion with 100 μg monoclonal antibody anti–VCAM-1. The results indicate that the enhanced adhesion of leukocytes to endothelium in wild-type mice was significantly reduced in ICAM-1
−/−
and completely abrogated in ICAM-1
−/−
/P-selectin
−/−
mice treated with ap IgG-APS compared with wild-type mice treated with ap IgG-APS (6.9±2.3, 0.4±0.4 versus 35±12, respectively). More importantly, this correlated with a significant reduction in thrombus size compared with wild-type mice treated with ap IgG-APS (895±259 μm
2
, 859±243 μm
2
versus 3816±672 μm
2
, respectively). Infusion of the mice with anti–VCAM-1 antibodies significantly reversed the enhanced adhesion of leukocytes (14.9±3 to 11.3±2.1) and thrombus size 3830±1008 μm
2
versus 876±548 μm
2
) in mice treated with ap IgG-APS. The data indicate that ICAM-1, P-selectin, and VCAM-1 expression are important in thrombotic complications by aPL antibodies and may provide novel targets for therapy in patients with APS.
LDL Downregulates CYP51 in Porcine Vascular Endothelial Cells and in the Arterial Wall Through a Sterol Regulatory Element Binding Protein-2–Dependent Mechanism
Authors: Rodríguez Cristina , Martínez-González José , Sánchez-Gómez Sonia , Badimon Lina
Published: June 2012

Abstract
—Hypercholesterolemia is associated with endothelial dysfunction and atherosclerotic lesion formation. By mRNA–differential display analysis, we have identified lanosterol 14α-demethylase (CYP51) as a gene highly regulated by native LDLs (nLDLs) in endothelial cells. CYP51 is a cytochrome P-450 enzyme involved in the postsqualene phases of cholesterol biosynthesis. CYP51 mRNA levels decrease in nLDL-treated cells in a dose- and time-dependent manner (9-fold after 24 hours with 180 mg of LDL cholesterol per deciliter), an effect that is blocked by cycloheximide. In parallel, sterol regulatory element (SRE) binding protein-2 (SREBP-2) expression falls (10-fold), without alteration in SREBP-1 level.
N
-Acetyl-leucyl-leucyl-norleucinal, which inhibits catabolism of the active form of SREBPs, abolished the effect of high concentrations of nLDL on CYP51 expression. Gel-shift assays performed with the SRE of the
cyp51
gene (
cyp51
-SRE) revealed a diminished SREBP-SRE interaction in LDL-treated cells. Moreover, nLDLs downregulate CYP51 promoter activity in transfection assays. Thus, atherogenic levels of nLDL downregulate endothelial CYP51 mRNA levels through a reduction in SRE–SREBP-2 interaction. Additionally, SREBP-2 and CYP51 mRNA levels are decreased in the arterial wall of hypercholesterolemic pigs. In summary, we have described for the first time, both in in vivo and in vitro systems, that CYP51 is expressed in the vascular wall and that it is downregulated together with SREBP-2 by high levels of nLDL. Because this transcription factor controls multiple cell lipid metabolism pathways, its regulation by nLDL could play a key role in lipid-mediated endothelial dysfunction.
AKAP-Mediated Targeting of Protein Kinase A Regulates Contractility in Cardiac Myocytes
Authors: Fink Mary A. , Zakhary Daniel R. , Mackey Julie A. , Desnoyer Russell W. , Apperson-Hansen Carolyn , Damron Derek S. , Bond Meredith
Published: June 2012

Abstract
—Compartmentalization of cAMP-dependent protein kinase A (PKA) by A-kinase anchoring proteins (AKAPs) targets PKA to distinct subcellular locations in many cell types. However, the question of whether AKAP-mediated PKA anchoring in the heart regulates cardiac contractile function has not been addressed. We disrupted AKAP-mediated PKA anchoring in cardiac myocytes by introducing, via adenovirus-mediated gene transfer, Ht31, a peptide that binds the PKA regulatory subunit type II (RII) with high affinity. This peptide competes with endogenous AKAPs for RII binding. Ht31P (a proline-substituted derivative), which does not bind RII, was used as a negative control. We then investigated the effects of Ht31 expression on RII distribution, Ca
2+
cycling, cell shortening, and PKA-dependent substrate phosphorylation. By confocal microscopy, we showed redistribution of RII from the perinuclear region and from periodic transverse striations in Ht31P-expressing cells to a diffuse cytosolic localization in Ht31-expressing cells. In the presence of 10 nmol/L isoproterenol, Ht31-expressing myocytes displayed an increased rate and amplitude of cell shortening and relaxation compared with control cells (uninfected and Ht31P-expressing myocytes); with isoproterenol stimulation we observed decreased time to 90% decline in Ca
2+
but no significant difference between Ht31-expressing and control cells in the rate of Ca
2+
cycling or amplitude of the Ca
2+
transient. The increase in PKA-dependent phosphorylation of troponin I and myosin binding protein C on isoproterenol stimulation was significantly reduced in Ht31-expressing cells compared with controls. Our results demonstrate that, in response to β-adrenergic stimulation, cardiomyocyte function and substrate phosphorylation by PKA is regulated by targeting of PKA by AKAPs.
Canine Ventricular Myocytes Possess a Renin-Angiotensin System That Is Upregulated With Heart Failure
Authors: Barlucchi Laura , Leri Annarosa , Dostal David E. , Fiordaliso Fabio , Tada Hideo , Hintze Thomas H. , Kajstura Jan , Nadal-Ginard Bernardo , Anversa Piero
Published: June 2012

Abstract
—Ventricular pacing leads to a dilated myopathy in which cell death and myocyte hypertrophy predominate. Because angiotensin II (Ang II) stimulates myocyte growth and triggers apoptosis, we tested whether canine myocytes express the components of the renin-angiotensin system (RAS) and whether the local RAS is upregulated with heart failure. p53 modulates transcription of angiotensinogen (Aogen) and AT
1
receptors in myocytes, raising the possibility that enhanced p53 function in the decompensated heart potentiates Ang II synthesis and Ang II–mediated responses. Therefore, the presence of mRNA transcripts for Aogen, renin, angiotensin-converting enzyme, chymase, and AT
1
and AT
2
receptors was evaluated by reverse transcriptase–polymerase chain reaction in myocytes. Changes in the protein expression of these genes were then determined by Western blot in myocytes from control dogs and dogs affected by congestive heart failure. p53 binding to the promoter of Aogen and AT
1
receptor was also determined. Ang II in myocytes was measured by ELISA and by immunocytochemistry and confocal microscopy. Myocytes expressed mRNAs for all the constituents of RAS, and heart failure was characterized by increased p53 DNA binding to Aogen and AT
1
. Additionally, protein levels of Aogen, renin, cathepsin D, angiotensin-converting enzyme, and AT
1
were markedly increased in paced myocytes. Conversely, chymase and AT
2
proteins were not altered. Ang II quantity and labeling of myocytes increased significantly with cardiac decompensation. In conclusion, dog myocytes synthesize Ang II, and activation of p53 function with ventricular pacing upregulates the myocyte RAS and the generation and secretion of Ang II. Ang II may promote myocyte growth and death, contributing to the development of heart failure.