bluuebunny/crossref_metadata_2025 huggingface dataset:
Regression of Hypertrophied Rat Pulmonary Arteries in Organ Culture Is Associated With Suppression of Proteolytic Activity, Inhibition of Tenascin-C, and Smooth Muscle Cell Apoptosis
Authors: Cowan Kyle Northcote , Jones Peter Lloyd , Rabinovitch Marlene
Published: June 2012

Abstract
—Increased elastase activity and deposition of the matrix glycoprotein tenascin-C (TN), codistributing with proliferating smooth muscle cells (SMCs), are features of pulmonary vascular disease. In pulmonary artery (PA) SMC cultures, TN is regulated by matrix metalloproteinases (MMPs) and mechanical stress. On attached collagen gels, MMPs upregulate TN, leading to SMC proliferation, whereas on floating collagen, reduced MMPs suppress TN and induce SMC apoptosis. We now investigate the response of SMCs in the whole vessel by comparing attached and floating conditions using either normal PAs derived from juvenile pigs or normal or hypertrophied rat PAs that were embedded in collagen gels for 8 days. Normal porcine PAs in attached collagen gels were characterized by increasing activity of MMP-2 and MMP-9 assessed by zymography and TN deposition detected by Western immunoblotting and densitometric analysis of immunoreactivity. PAs on floating collagen showed reduced activity of both MMPs and deposition of TN. Tenascin-rich foci were associated with proliferating cell nuclear antigen immunoreactivity, and TN-poor areas with apoptosis, by terminal deoxynucleotidyl transferase–mediated nick end labeling assay, but no difference in wall thickness was observed. Although normal rat PAs were similar to piglet vessels, hypertrophied rat PAs showed an amplified response. Increased elastase, MMP-2, TN, and elastin deposition, as well as SMC proliferating cell nuclear antigen positivity, correlated with progressive medial thickening on attached collagen, whereas reduced MMP-2, elastase, TN, and induction of SMC apoptosis accompanied regression of the thickened media on floating collagen. In showing that hypertrophied SMCs in the intact vessel can be made to apoptose and that resorption of extracellular matrix can be achieved by inhibition of elastase and MMPs, our study suggests novel strategies to reverse vascular disease.
Direct Demonstration of P-Selectin– and VCAM-1–Dependent Mononuclear Cell Rolling in Early Atherosclerotic Lesions of Apolipoprotein E–Deficient Mice
Authors: Ramos Carroll L. , Huo Yuqing , Jung Unsu , Ghosh Shukti , Manka David R. , Sarembock Ian J. , Ley Klaus
Published: June 2012

Abstract
—Apolipoprotein E–deficient (ApoE
−/−
) mice develop atherosclerotic lesions throughout the arterial tree, including the carotid bifurcation. Although the expression of adhesion molecules such as ICAM-1, vascular cell adhesion molecule-1 (VCAM-1), and P-selectin on endothelium that overlie atherosclerotic plaques has been implicated in monocyte recruitment to developing lesions, monocyte adhesion in atherosclerotic vessels has not been observed directly. To investigate which adhesion molecules may be important in monocyte adhesion to atherosclerotic lesions, an isolated mouse carotid artery preparation was developed and perfused with mononuclear cells. We show rolling and attachment of the human monocytic cell line U937 and the mouse monocyte-macrophage cell line P388D1 in carotid arteries from 10- to 12-week-old ApoE
−/−
and C57BL/6 wild-type mice fed a Western-type diet (21% fat wt/wt) for 4 to 5 weeks. No rolling was observed in carotid arteries from C57BL/6 or BALB/c wild-type mice fed a chow diet and little was observed in BALB/c mice fed a Western-type diet. This model represents early lesion development as shown by minimal macrophage infiltration in the intima of carotid arteries from ApoE
−/−
mice fed a Western-type diet. Rolling was observed at shear stresses that were characteristic of the low-shear recirculation zone near the carotid bifurcation. Mononuclear cell attachment and rolling were significantly inhibited by monoclonal antibody blockade of P-selectin or its leukocyte ligand P-selectin glycoprotein ligand-1. Rolling velocities increased after monoclonal antibody blockade of mononuclear cell α
4
-integrin or VCAM-1, which indicates that α
4
-integrin interacting with VCAM-1 stabilizes rolling interactions and prolongs monocyte transit times.
Modulation of Iron Uptake in Heart by L-Type Ca
2+
Channel Modifiers
Authors: Tsushima Robert G. , Wickenden Alan D. , Bouchard Ron A. , Oudit Gavin Y. , Liu Peter P. , Backx Peter H.
Published: June 2012

Abstract
—Heart failure is the leading cause of mortality in patients with transfusional iron (Fe) overload in which myocardial iron uptake ensues via a transferrin-independent process. We examined the ability of L-type Ca
2+
channel modifiers to alter Fe
2+
uptake by isolated rat hearts and ventricular myocytes. Perfusion of rat hearts with 100 nmol/L
59
Fe
2+
and 5 mmol/L ascorbate resulted in specific
59
Fe
2+
uptake of 20.4±1.9 ng of Fe per gram dry wt. Abolishing myocardial electrical excitability with 20 mmol/L KCl reduced specific
59
Fe
2+
uptake by 60±7% (
P
<0.01), which suggested that a component of myocardial Fe
2+
uptake depends on membrane voltage. Accordingly,
59
Fe
2+
uptake was inhibited by 10 μmol/L nifedipine (45±12%,
P
<0.02) and 100 μmol/L Cd
2+
(86±3%;
P
<0.001) while being augmented by 100 nmol/L Bay K 8644 (61±18%,
P
<0.01) or 100 nmol/L isoproterenol (40±12%,
P
<0.05). By contrast, uptake of 100 nmol/L ferric iron (
59
Fe
3+
) was significantly lower (1.4±0.3 ng Fe per gram dry wt;
P
<0.001) compared with divalent iron. These data suggest that a component of Fe
2+
uptake into heart occurs via the L-type Ca
2+
channel in myocytes. To investigate this further, the effects of Fe
2+
on cardiac myocyte L-type Ca
2+
currents were measured. In the absence of Ca
2+
, noninactivating nitrendipine-sensitive Fe
2+
currents were recorded with 15 mmol/L [Fe
2+
]
o
. Low concentrations of Fe
2+
enhanced Ca
2+
current amplitude and slowed inactivation rates, which was consistent with Fe
2+
entry into the cell, whereas higher Fe
2+
levels caused dose-dependent decreases in peak current. Fe
3+
had no effect on current amplitude or decay. Combined, our data suggest that myocardial Fe
2+
uptake occurs via L-type Ca
2+
channels and that blockade of these channels might be useful in the treatment of patients with excessive serum iron levels.
The Angiotensin II–Dependent Association of Jak2 and c-Src Requires the N-Terminus of Jak2 and the SH2 Domain of c-Src
Authors: Sayeski Peter P. , Ali M. Showkat , Hawks Kim , Frank Stuart J. , Bernstein Kenneth E.
Published: June 2012

Abstract
—The binding of angiotensin II (Ang II) to AT
1
is known to increase the kinase activity of several nonreceptor tyrosine kinases including Jak2 and c-Src. In the present study, we demonstrate that treatment of vascular smooth muscle cells with Ang II results in a rapid and transient association of Jak2 and c-Src. This association is dependent on a catalytically active Jak2 kinase, because it is blocked both by pharmacological means and by the inability of a catalytically inactive Jak2 to associate with c-Src. c-Src bound tyrosine phosphorylated Jak2 but was unable to bind an equal amount of unphosphorylated Jak2 protein, indicating that the SH2 domain of c-Src mediates this association. In vivo studies indicated that c-Src binds the N-terminus of Jak2 as expression of a Jak2 molecule lacking the initial 240 amino acids, including 16 tyrosines, and was unable to bind c-Src. Lastly, using transiently transfected COS-7 cells, we found that Ang II treatment induced an association between c-Src and wild-type Jak2 but not between c-Src and the Jak2 molecule that lacks the initial 240 amino acids. Thus, our data suggest that in addition to increasing the kinase activities Jak2 and c-Src, treatment of cells with Ang II results in the physical association of Jak2 and c-Src; an association that is mediated by the SH2 domain of c-Src and the N-terminus of Jak2.
Downregulation of Connexin 45 Gene Products During Mouse Heart Development
Authors: Alcoléa Sébastien , Théveniau-Ruissy Magali , Jarry-Guichard Thérèse , Marics Irène , Tzouanacou Elena , Chauvin Jean-Paul , Briand Jean-Paul , Moorman Antoon F. M. , Lamers Wouter H. , Gros Daniel B.
Published: June 2012

Abstract
—The electrical activity in heart is generated in the sinoatrial node and then propagates to the atrial and ventricular tissues. The gap junction channels that couple the myocytes are responsible for this propagation process. The gap junction channels are dodecamers of transmembrane proteins of the connexin (Cx) family. Three members of this family have been demonstrated to be synthesized in the cardiomyocytes: Cx40, Cx43, and Cx45. In addition, each of them has been shown to form channels with unique and specific electrophysiological properties. Understanding the conduction phenomenon requires detailed knowledge of the spatiotemporal expression pattern of these Cxs in heart. The expression patterns of Cx40 and Cx43 have been previously described in the adult heart and during its development. Here we report the expression of Cx45 gene products in mouse heart from the stage of the first contractions (8.5 days postcoitum [dpc]) to the adult stage. The Cx45 gene transcript was demonstrated by reverse transcriptase–polymerase chain reaction experiments to be present in heart at all stages investigated. Between 8.5 and 10.5 dpc it was shown by in situ hybridization to be expressed in low amounts in all cardiac compartments (including the inflow and outflow tracts and the atrioventricular canal) and then to be downregulated from 11 to 12 dpc onward. At subsequent fetal stages, the transcript was weakly detected in the ventricles, with the most distinct expression in the outflow tract. Cx45 protein was demonstrated by immunofluorescence microscopy to be expressed in the myocytes of young embryonic hearts (8.5 to 9.5 dpc). However, beyond 10.5 dpc the protein was no longer detected with this technique in the embryonic, fetal, or neonatal working myocardium, although it could be shown by immunoblotting that the protein was still synthesized in neonatal heart. In the major part of adult heart, Cx45 was undetectable. It was, however, clearly seen in the anterior regions of the interventricular septum and in trace amounts in some small foci dispersed in the ventricular free walls. Cx45 gene is the first Cx gene so far demonstrated to be activated in heart at the stage of the first contractions. The coordination of myocytes during the slow peristaltic contractions that occur at this stage would thus appear to be controlled by the Cx45 channels.
Nitric Oxide/cAMP Interactions in the Control of Rat Renal Vascular Resistance
Authors: Sandner Peter , Kornfeld Mark , Ruan Xiaoping , Arendshorst William J. , Kurtz Armin
Published: June 2012

Abstract
—This study aimed to characterize the interaction between nitric oxide (NO)- and cAMP-related pathways in the control of renal blood flow. Using the isolated perfused rat kidney model, we determined the effects of inhibition of NO formation by
N
ω
-nitro-
l
-arginine methyl ester (L-NAME; 1 mmol/L) and of NO administration by sodium nitroprusside (SNP, 10 μmol/L) on renal vascular resistance under conditions of elevated vascular cAMP levels. cAMP levels were increased either by adenylate cyclase activation via isoproterenol or by inhibition of cAMP phosphodiesterases (PDEs) 1, 3, and 4. We found that L-NAME markedly increased vascular resistance and that this effect was completely reversed by SNP. Both isoproterenol and inhibitors of the cAMP PDEs lowered basal vascular resistance. In the presence of isoproterenol (3 nmol/L) and inhibitors of PDE-1 [8-methoxymethyl-l-methyl-3-(2-methylpropyl)-xanthine; 8-MM-IBMX, 20 μmol/L] and PDE-4 (rolipram, 20 μmol/L), L-NAME again substantially increased vascular resistance, and this effect of L-NAME was completely reversed by SNP. In the presence of the PDE-3 inhibitors milrinone (20 μmol/L) and trequinsin (200 nmol/L), however, both L-NAME and SNP failed to exert any additional effects. Because PDE-3 is a cGMP-inhibited cAMP PDE and because the vasodilatory effect of SNP was abrogated by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) (20 μmol/L), our findings are compatible with the idea that an action of NO on PDE-3 could account for the vasodilatory properties of NO on the renal vasculature. Moreover, our findings suggest that PDE-3 activity is an important determinant of renal vascular resistance.
Nitric Oxide Inhibits Capacitative Cation Influx in Human Platelets by Promoting Sarcoplasmic/Endoplasmic Reticulum Ca
2+
-ATPase–Dependent Refilling of Ca
2+
Stores
Authors: Trepakova Elena S. , Cohen Richard A. , Bolotina Victoria M.
Published: June 2012

Abstract
—Nitric oxide (NO) is a potent inhibitor of thrombin-induced increase in cytoplasmic free Ca
2+
concentration and aggregation in platelets, but the precise mechanism of this inhibition is unclear. To measure Ca
2+
/Mn
2+
influx in intact platelets and to monitor Ca
2+
uptake into the stores in permeabilized platelets, fura-2 was used. In intact platelets, maximal capacitative Ca
2+
and Mn
2+
influx developed rapidly (within 30 s) after fast release of Ca
2+
from the stores with thrombin (0.5 U/mL) or slowly (within 5 to 10 minutes) following passive Ca
2+
leak caused by inhibition of sarcoplasmic/endoplasmic reticulum Ca
2+
-ATPase (SERCA) with 30 μmol/L 2,5-di-(
tert
-butyl)-1,4-benzohydroquinone (BHQ). NO (1 μmol/L) inhibited capacitative Ca
2+
and Mn
2+
influx independently of the time after thrombin application. In contrast, the effect of NO on BHQ-induced Ca
2+
and Mn
2+
influx was observed only during the first few minutes after BHQ application and completely disappeared when capacitative cation influx reached its maximum. In Ca
2+
-free medium, NO reduced the peak Ca
2+
rise caused by thrombin and significantly promoted Ca
2+
back-sequestration into the stores. Both effects disappeared in the presence of BHQ. Inhibition of guanylate cyclase with H-(1,2,4) oxadiazolo(4,3-a) quinoxallin-1-one (10 μmol/L) attenuated but did not prevent the effects of NO on cytoplasmic free Ca
2+
concentration. Inhibition of Ca
2+
uptake by mitochondria did not change the effects of NO. In permeabilized platelets, NO accelerated back-sequestration of Ca
2+
into the stores after inositol-1,4,5-trisphosphate–induced Ca
2+
release or after addition of Ca
2+
(1 μmol/L) in the absence of inositol-1,4,5-trisphosphate. The effect of NO depended on the initial rate of Ca
2+
uptake and on the concentration of ATP and was abolished by BHQ, indicating the direct involvement of SERCA. These data strongly support the hypothesis that NO inhibits store-operated cation influx in human platelets indirectly via acceleration of SERCA-dependent refilling of Ca
2+
stores.
Endothelial Cells Modulate the Proliferation of Mural Cell Precursors via Platelet-Derived Growth Factor-BB and Heterotypic Cell Contact
Authors: Hirschi Karen K. , Rohovsky Stephanie A. , Beck Laurence H. , Smith Sandra R. , D’Amore Patricia A.
Published: June 2012

Abstract
—Embryological data suggest that endothelial cells (ECs) direct the recruitment and differentiation of mural cell precursors. We have developed in vitro coculture systems to model some of these events and have shown that ECs direct the migration of undifferentiated mesenchymal cells (10T1/2 cells) and induce their differentiation toward a smooth muscle cell/pericyte lineage. The present study was undertaken to investigate cell proliferation in these cocultures. ECs and 10T1/2 cells were cocultured in an underagarose assay in the absence of contact. There was a 2-fold increase in bromodeoxyuridine labeling of 10T1/2 cells in response to ECs, which was completely inhibited by the inclusion of neutralizing antiserum against platelet-derived growth factor (PDGF)-B. Antisera against PDGF-A, basic fibroblast growth factor, or transforming growth factor (TGF)-β had no effect on EC-stimulated 10T1/2 cell proliferation. EC proliferation was not influenced by coculture with 10T1/2 cells in the absence of contact. The cells were then cocultured so that contact was permitted. Double labeling and fluorescence-activated cell sorter analysis revealed that ECs and 10T1/2 cells were growth-inhibited by 43% and 47%, respectively. Conditioned media from contacting EC-10T1/2 cell cocultures inhibited the growth of both cell types by 61% and 48%, respectively. Although we have previously shown a role for TGF-β in coculture-induced mural cell differentiation, growth inhibition resulting from contacting cocultures or conditioned media was not suppressed by the presence of neutralizing antiserum against TGF-β. Furthermore, the decreased proliferation of 10T1/2 cells in the direct cocultures could not be attributed to downregulation of the PDGF-B in ECs or the PDGF receptor-β in the 10T1/2 cells. Our data suggest that modulation of proliferation occurs during EC recruitment of mesenchymal cells and that heterotypic cell-cell contact and soluble factors play a role in growth control during vessel assembly.
Endothelial Implants Inhibit Intimal Hyperplasia After Porcine Angioplasty
Authors: Nugent Helen M. , Rogers Campbell , Edelman Elazer R.
Published: June 2012

Abstract
—The perivascular implantation of tissue-engineered endothelial cells around injured arteries offers an opportunity to study fundamental vascular physiology as well as restore and improve tissue function. Cell source is an important issue because the ability to implant either xenogeneic or allogeneic cells would greatly enhance the clinical applications of tissue-engineered grafts. We investigated the biological and immunological responses to endothelial cell xenografts and allografts in pigs 4 weeks after angioplasty of the carotid arteries. Porcine or bovine aortic endothelial cells were cultured within Gelfoam matrices and implanted in the perivascular space of 42 injured arteries. Both porcine and bovine endothelial cell grafts reduced the restenosis index compared with control by 54% and 46%, respectively. Perivascular heparin release devices, formulated to release heparin at twice the rate of release of heparan sulfate proteoglycan from endothelial cell implants, produced no significant reduction in the restenosis index. Endothelial cell implants also reduced occlusive thrombosis compared with control and heparin release devices. Host immune responses to endothelial implants were investigated by immunohistochemical examination of explanted devices and by immunocytochemistry of serum samples. The bovine cell grafts displayed infiltration of leukocytes, consisting primarily of lymphocytes, and caused an increase in antibodies detected in serum samples. Reduced cellular infiltration and no humoral response were detected in animals that received allografts. Despite the difference in immune response, the biological effects of xenografts or allografts did not differ significantly.
Tumor Necrosis Factor-α Contributes to Ischemia- and Reperfusion-Induced Endothelial Activation in Isolated Hearts
Authors: Kupatt Christian , Habazettl Helmut , Goedecke Axel , Wolf Dieter A. , Zahler Stefan , Boekstegers Peter , Kelly Ralph A. , Becker Bernhard F.
Published: June 2012

Abstract
—During myocardial reperfusion, polymorphonuclear neutrophil (PMN) adhesion involving the intercellular adhesion molecule-1 (ICAM-1) may lead to aggravation and prolongation of reperfusion injury. We studied the role of early tumor necrosis factor-α (TNF-α) cleavage and nuclear factor-κB (NF-κB) activation on ICAM-1 expression and venular adhesion of PMN in isolated hearts after ischemia (15 minutes) and reperfusion (30 to 480 minutes). NF-κB activation (electromobility shift assay) was found after 30 minutes of reperfusion and up to 240 minutes. ICAM-1 mRNA, assessed by Northern blot, increased during the same interval. Functional effect of newly synthesized adhesion molecules was found by quantification (in situ fluorescence microscopy) of PMN, given as bolus after ischemia, which became adherent to small coronary venules (10 to 50 μm in diameter). After 480 minutes of reperfusion, ICAM-1–dependent PMN adhesion increased 2.5-fold compared with PMN adhesion obtained during acute reperfusion. To study the influence of NF-κB on PMN adhesion, we inhibited NF-κB activation by transfection of NF-κB decoy oligonucleotides into isolated hearts using HJV-liposomes. Decoy NF-κB but not control oligonucleotides blocked ICAM-1 upregulation and inhibited the subacute increase in PMN adhesion. Similar effects were obtained using BB 1101 (10 μg), an inhibitor of TNF-α cleavage enzyme. These data suggest that ischemia and reperfusion in isolated hearts cause liberation of TNF-α, activation of NF-κB, and upregulation of ICAM-1, an adhesion molecule involved in inflammatory response after ischemia and reperfusion.
The Sarcoplasmic Reticulum and the Na
+
/Ca
2+
Exchanger Both Contribute to the Ca
2+
Transient of Failing Human Ventricular Myocytes
Authors: Dipla Konstantina , Mattiello Julian A. , Margulies Kenneth B. , Jeevanandam Valluvan , Houser Steven R.
Published: June 2012

Abstract
—Our objective was to determine the respective roles of the sarcoplasmic reticulum (SR) and the Na
+
/Ca
2+
exchanger in the small, slowly decaying Ca
2+
transients of failing human ventricular myocytes. Left ventricular myocytes were isolated from explanted hearts of patients with severe heart failure (n=18). Cytosolic Ca
2+
, contraction, and action potentials were measured by using indo-1, edge detection, and patch pipettes, respectively. Selective inhibitors of SR Ca
2+
transport (thapsigargin) and reverse-mode Na
+
/Ca
2+
exchange activity (No. 7943, Kanebo Ltd) were used to define the respective contribution of these processes to the Ca
2+
transient. Ca
2+
transients and contractions induced by action potentials (AP transients) at 0.5 Hz exhibited phasic and tonic components. The duration of the tonic component was determined by the action potential duration. Ca
2+
transients induced by caffeine (Caf transients) exhibited only a phasic component with a rapid rate of decay that was dependent on extracellular Na
+
. The SR Ca
2+
-ATPase inhibitor thapsigargin abolished the phasic component of the AP Ca
2+
transient and of the Caf transient but had no significant effect on the tonic component of the AP transient. The Na
+
/Ca
2+
exchange inhibitor No. 7943 eliminated the tonic component of the AP transient and reduced the magnitude of the phasic component. In failing human myocytes, Ca
2+
transients and contractions exhibit an SR-related, phasic component and a slow, reverse-mode Na
+
/Ca
2+
exchange–related tonic component. These findings suggest that Ca
2+
influx via reverse-mode Na
+
/Ca
2+
exchange during the action potential may contribute to the slow decay of the Ca
2+
transient in failing human myocytes.
Rho Family Small G Proteins Play Critical Roles in Mechanical Stress–Induced Hypertrophic Responses in Cardiac Myocytes
Authors: Aikawa Ryuichi , Komuro Issei , Yamazaki Tsutomu , Zou Yunzeng , Kudoh Sumiyo , Zhu Weidong , Kadowaki Takashi , Yazaki Yoshio
Published: June 2012

Abstract
—Mechanical stress induces a variety of hypertrophic responses, such as activation of protein kinases, reprogramming of gene expression, and an increase in protein synthesis. In the present study, to elucidate how mechanical stress induces such events, we examined the role of Rho family small GTP-binding proteins (G proteins) in mechanical stress–induced cardiac hypertrophy. Treatment of neonatal rat cardiomyocytes with the C3 exoenzyme, which abrogates Rho functions, suppressed stretch-induced activation of extracellular signal–regulated protein kinases (ERKs). Overexpression of the Rho GDP dissociation inhibitor (Rho-GDI), dominant-negative mutants of RhoA (DNRhoA), or DNRac1 significantly inhibited stretch-induced activation of transfected ERK2. Overexpression of constitutively active mutants of RhoA slightly activated ERK2 in cardiac myocytes. Overexpression of
C
-terminal Src kinase, which inhibits functions of the Src family of tyrosine kinases, or overexpression of DNRas had no effect on stretch-induced activation of transfected ERK2. The promoter activity of skeletal α-actin and c-
fos
genes was increased by stretch, and these increases were completely inhibited by either cotransfection of Rho-GDI or pretreatment with C3 exoenzyme. Mechanical stretch increased phenylalanine incorporation into cardiac myocytes by ≈1.5-fold compared with control, and this increase was also significantly suppressed by pretreatment with C3 exoenzyme. Overexpression of Rho-GDI or DNRhoA did not affect angiotensin II–induced activation of ERK. ERKs were activated by culture media conditioned by stretch of cardiomyocytes without any treatment, but not of cardiomyocytes with pretreatment by C3 exoenzyme. These results suggest that the Rho family of small G proteins plays critical roles in mechanical stress–induced hypertrophic responses.
α-Adrenergic Stimulation Mediates Glucose Uptake Through Phosphatidylinositol 3-Kinase in Rat Heart
Authors: Doenst Torsten , Taegtmeyer Heinrich
Published: June 2012

Abstract
—We examined whether insulin and catecholamines share common pathways for their stimulating effects on glucose uptake. We perfused isolated working rat hearts with Krebs-Henseleit buffer containing [2-
3
H]glucose (5 mmol/L, 0.05 μCi/mL) and sodium oleate (0.4 mmol/L). In the absence or presence of the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin (3 μmol/L), we added insulin (1 mU/mL), epinephrine (1 μmol/L), phenylephrine (100 μmol/L) plus propranolol (10 μmol/L, selective α-adrenergic stimulation), or isoproterenol (1 μmol/L) plus phentolamine (10 μmol/L, selective β-adrenergic stimulation) to the perfusate. Cardiac power was found to be stable in all groups (between 8.07±0.68 and 10.7±0.88 mW) and increased (25% to 47%) with addition of epinephrine, but not with selective α- and β-adrenergic stimulation. Insulin and epinephrine, as well as selective α- and β-receptor stimulation, increased glucose uptake (the following values are in μmol/[min · g dry weight]: basal, 1.19±0.13; insulin, 3.89±0.36; epinephrine, 3.46±0.27; α-stimulation, 4.08±0.40; and β-stimulation, 3.72±0.34). Wortmannin completely inhibited insulin-stimulated and selective α-stimulated glucose uptake, but it did not affect the epinephrine-stimulated or selective β-stimulated glucose uptake. Sequential addition of insulin and epinephrine or insulin and α-selective stimulation showed additive effects on glucose uptake in both cases. Wortmannin further blocked the effects of insulin on glycogen synthesis. We conclude that α-adrenergic stimulation mediates glucose uptake in rat heart through a PI3-K–dependent pathway. However, the additive effects of α-adrenergic stimulation and insulin suggest 2 different isoforms of PI3-K, compartmentation of PI3-K, potentiation, or inhibition by wortmannin of another intermediate of the α-adrenergic signaling cascade. The stimulating effects of both the α- and the β-adrenergic pathways on glucose uptake are independent of changes in cardiac performance.
TIMP-4 Is Regulated by Vascular Injury in Rats
Authors: Dollery Clare M. , McEwan Jean R. , Wang Mingsheng , Sang Qingxiang Amy , Liu Yiliang E. , Shi Y. Eric
Published: June 2012

Abstract
—The role of basement membrane–degrading matrix metalloproteinases (MMPs) in enabling vascular smooth muscle cell migration after vascular injury has been established in several animal models. In contrast, the role of their native inhibitors, the tissue inhibitors of matrix metalloproteinases (TIMPs), has remained unproven despite frequent coregulation of MMPs and TIMPs in other disease states. We have investigated the time course of expression and localization of TIMP-4 in rat carotid arteries 6 hours, 24 hours, 3 days, 7 days, and 14 days after balloon injury by in situ hybridization, immunohistochemistry, and Western blot analysis. TIMP-4 protein was present in the adventitia of injured carotid arteries from 24 hours after injury. At 7 and 14 days after injury, widespread immunostaining for TIMP-4 was observed throughout the neointima, media, and adventitia of injured arteries. Western blot analysis confirmed the quantitative increase in TIMP-4 protein at 7 and 14 days. In situ hybridization detected increased expression of TIMP-4 as early as 24 hours after injury and a marked induction in neointimal cells 7 days after injury. We then studied the effect of TIMP-4 protein on the migration of smooth muscle cells through a matrix-coated membrane in vitro and demonstrated a 53% reduction in invasion of rat vascular smooth muscle cells. These data and the temporal relationship between the upregulation of TIMP-4, its accumulation, and the onset of collagen deposition suggest an important role for TIMP-4 in the proteolytic balance of the vasculature controlling both smooth muscle migration and collagen accumulation in the injured arterial wall.
Mitogen-Activated Protein/Extracellular Signal–Regulated Kinase Inhibition Attenuates Angiotensin II–Mediated Signaling and Contraction in Spontaneously Hypertensive Rat Vascular Smooth Muscle Cells
Authors: Touyz R. M. , El Mabrouk M. , He G. , Wu X-H. , Schiffrin E. L.
Published: June 2012

Abstract
—This study investigates the role of extracellular signal–regulated kinases (ERKs) in angiotensin II (Ang II)–generated intracellular second messengers (cytosolic free Ca
2+
concentration, ie, [Ca
2+
]
i
, and pH
i
) and in contraction in isolated vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and control Wistar Kyoto rats (WKY) using the selective mitogen-activated protein (MAP)/ERK inhibitor, PD98059. VSMCs from mesenteric arteries were cultured on Matrigel basement membrane matrix. These cells, which exhibit a contractile phenotype, were used to measure [Ca
2+
]
i
, pH
i
, and contractile responses to Ang II (10
–12
to 10
–6
mol/L) in the absence and presence of PD98059 (10
–5
mol/L). [Ca
2+
]
i
and pH
i
were measured by fura-2 and BCECF methodology, respectively, and contraction was determined by photomicroscopy. Ang II–stimulated ERK activity was measured by Western blot analysis using a phospho-specific ERK-1/ERK-2 antibody and by an MAPK enzyme assay. Ang II increased [Ca
2+
]
i
and pH
i
and contracted cells in a dose-dependent manner. Maximum Ang II–elicited contraction was greater (
P
<0.05) in SHR (41.9±5.1% reduction in cell length relative to basal length) than in WKY (28.1±3.0% reduction in cell length relative to basal length). Basal [Ca
2+
]
i
, but not basal pH
i
, was higher in SHR compared with WKY. [Ca
2+
]
i
and pH
i
effects of Ang II were enhanced (
P
<0.05) in SHR compared with WKY (maximum Ang II–induced response [E
max
] of [Ca
2+
]
i
, 576±24 versus 413±43 nmol/L; E
max
of pH
i
, 7.33±0.01 versus 7.27±0.03, SHR versus WKY). PD98059 decreased the magnitude of contraction and attenuated the augmented Ang II–elicited contractile responses in SHR (E
max
,19.3±3% reduction in cell length relative to basal length). Ang II–stimulated [Ca
2+
]
i
(E
max
, 294±55 nmol/L) and pH
i
(E
max
, 7.27±0.04) effects were significantly reduced by PD98059 in SHR. Ang II–induced ERK activity was significantly greater (
P
<0.05) in SHR than in WKY. In conclusion, Ang II–stimulated signal transduction and associated VSMC contraction are enhanced in SHR. MAP/ERK inhibition abrogated sustained contraction and normalized Ang II effects in SHR. These data suggest that ERK-dependent signaling pathways influence contraction and that they play a role in vascular hyperresponsiveness in SHR.
Molecular Mechanisms of Endothelin-1–Induced Cell-Cycle Progression
Authors: Suzuki Etsu , Nagata Daisuke , Kakoki Masao , Hayakawa Hiroshi , Goto Atsuro , Omata Masao , Hirata Yasunobu
Published: June 2012

Abstract
—Although it is well established that endothelin-1 (ET-1) has not only vasoconstrictive effects but also mitogenic effects, which seem to be implicated in vascular remodeling, little is known about the molecular mechanisms by which ET-1 induces cell-cycle progression. In this study, we examined the effects of ET-1 on the cell-cycle regulatory machinery, including cyclins, cyclin-dependent kinase (cdk), and cdk inhibitors in NIH3T3 cells. ET-1 increased cyclin D1 protein (5.1±1.9-fold increase, 8 hours after stimulation,
P
<0.05), cdk4 kinase activity (2.8±0. 5-fold increase, 12 hours after stimulation,
P
<0.01), and cdk2 kinase activity (2.1±0.4-fold increase, 16 hours after stimulation,
P
<0.05) in a time- and dose-dependent manner. ET-1–induced increase in cyclin D1 protein, and cdk4 kinase activity was not significantly inhibited by an inhibitor of the mitogen-activated protein kinase kinase 1/2, PD98059, nor by the protein kinase C inhibitor calphostin C, whereas ET-1–induced upregulation of cyclin D1 protein and cdk4 kinase activity was significantly inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002. In contrast, ET-1–induced activation of cdk2 kinase was significantly inhibited by PD98059, calphostin C, and LY294002. ET-1 increased
3
H-thymidine uptake in a time-dependent fashion (0 hours, 4216±264 cpm per well; 8 hours, 5025±197 cpm per well; 16 hours, 9239±79 cpm per well,
P
<0.001 versus 0 hours). ET-1–induced increase in
3
H-thymidine uptake was significantly inhibited by PD98059, calphostin C, and LY294002. These results suggest that ET-1–induced cell-cycle progression is, at least in part, mediated by the extracellular signal-regulated kinase, protein kinase C, and phosphatidylinositol 3-kinase and that those pathways may be involved in the progression of the cell cycle at distinct points.
Identification and Expression of δ-Isoforms of the Multifunctional Ca
2+
/Calmodulin-Dependent Protein Kinase in Failing and Nonfailing Human Myocardium
Authors: Hoch Brigitte , Meyer Rudolf , Hetzer Roland , Krause Ernst-Georg , Karczewski Peter
Published: June 2012

Abstract
—Despite its importance for the regulation of heart function, little is known about the isoform expression of the multifunctional Ca
2+
/calmodulin-dependent protein kinase (CaMKII) in human myocardium. In this study, we investigated the spectrum of CaMKII isoforms δ
2
, δ
3
, δ
4
, δ
8
, and δ
9
in human striated muscle tissue. Isoform δ
3
is characteristically expressed in cardiac muscle. In skeletal muscle, specific expression of a new isoform termed δ
11
is demonstrated. Complete sequencing of human δ
2
cDNA, representing all common features of the investigated CaMKII subclass, revealed its high homology to the corresponding rat cDNA. Comparative semiquantitative reverse transcription–polymerase chain reaction analyses from left ventricular tissues of normal hearts and from patients suffering from dilated cardiomyopathy showed a significant increase in transcript levels of isoform δ
3
relative to the expression of glyceraldehyde-3-phosphate dehydrogenase in diseased hearts (101.6±11.0% versus 64.9±9.9% in the nonfailing group;
P
<0.05, n=6). Transcript levels of the other investigated cardiac CaMKII isoforms remained unchanged. At the protein level, by using a subclass-specific antibody, we observed a similar increase of a δ-CaMKII–specific signal (7.2±1.0 versus 3.8±0.7 optical density units in the nonfailing group;
P
<0.05, n=4 through 6). The diseased state of the failing hearts was confirmed by a significant increase in transcript levels for atrial natriuretic peptide (292.9±76.4% versus 40.1±3.2% in the nonfailing group;
P
<0.05, n=3 through 6). Our data characterize for the first time the δ-CaMKII isoform expression pattern in human hearts and demonstrate changes in this expression pattern in heart failure.
Failure of Calcineurin Inhibitors to Prevent Pressure-Overload Left Ventricular Hypertrophy in Rats
Authors: Zhang Weiguo , Kowal Robert C. , Rusnak Frank , Sikkink Robert A. , Olson Eric N. , Victor Ronald G.
Published: June 2012

Abstract
—A rapidly emerging body of literature implicates a pivotal role for the Ca
2+
-calmodulin–dependent phosphatase calcineurin as a cellular target for a variety of Ca
2+
-dependent signaling pathways culminating in left ventricular hypertrophy (LVH). Most of the recent experimental support for this hypothesis is derived from in vitro studies or in vivo studies in transgenic mice expressing activated calcineurin or mutant sarcomeric proteins. The aim of the present study was to test whether calcineurin inhibitors, cyclosporin A (CsA) and FK 506, prevent pressure-overload LVH using 2 standard rat models: (1) the spontaneously hypertensive rat (SHR) and (2) aortic banding. The major new findings are 2-fold. First, in SHR, LVH (left ventricular weight to body weight ratio) was unaffected by a dose of CsA (5 mg · kg
−1
· d
−1
) that was sufficient to raise blood pressure and to inhibit calcineurin-mediated transcriptional activation in skeletal muscle. Second, in rats with aortic banding, LVH was unaffected by FK 506 (0.3 mg · kg
−1
· d
−1
) or even higher doses of CsA (10 and 20 mg · kg
−1
· d
−1
) that were sufficient to inhibit 90% of total calcineurin phosphatase activity in the hypertrophied myocardium. In the latter experiments, CsA blocked neither the elevated left ventricular end-diastolic pressures, a measure of diastolic function, nor the induction in atrial natriuretic peptide mRNA in the hypertrophic ventricles. Thus, in numerous experiments, systemic administration of potent calcineurin inhibitors did not prevent the development of LVH in 2 classic models of pressure-overload hypertrophy. These results demonstrate that pressure-overload hypertrophy can arise through calcineurin-independent pathways.
Cyclosporine Attenuates Pressure-Overload Hypertrophy in Mice While Enhancing Susceptibility to Decompensation and Heart Failure
Authors: Meguro Tomomi , Hong Chull , Asai Kuniya , Takagi Gen , McKinsey Timothy A. , Olson Eric N. , Vatner Stephen F.
Published: June 2012

Abstract
—Left ventricular hypertrophy (LVH) is a compensatory mechanism to cope with pressure overload. Recently, a calcineurin pathway mediating LVH and its prevention by cyclosporine was reported. We examined whether calcineurin mediates LVH due to pressure overload in mice. Pressure overload was induced by aortic banding in 53 mice (32 treated with cyclosporine [25 mg · kg
−1
· d
−1
], 21 treated with vehicle). There were 17 sham-operated mice (9 treated with vehicle, 8 treated with cyclosporine). At 3 weeks after surgery, LV weight to body weight was greater in the nontreatment banded group (4.39±0.16 mg/g) than in the cyclosporine-treated banded group (3.95±0.14 mg/g,
P
<0.05), with both groups being greater compared with the entire group of sham-operated mice (3.02±0.04 mg/g). The pressure gradient between the ascending and abdominal aorta was not different between the cyclosporine-treated (49.6±6.1 mm Hg) and nontreatment groups (48.7±4.6 mm Hg). Although LV systolic pressure was lower in the cyclosporine-treated banded animals, LV systolic wall stress was similar in the nontreatment banded group and in the cyclosporine-treated group. However, LV dP/dt was lower (
P
=0.05) in the cyclosporine-treated banded group (4774±656 mm Hg/s) than in the nontreatment banded group (6604±516 mm Hg/s). During the protocol, 23 of 32 mice in the cyclosporine-treated group and 9 of 21 mice in the nontreatment group died. All deaths occurred within 10 days after surgery. Deaths caused by heart failure were 7.2-fold higher (
P
<0.05) in the cyclosporine-treated group, whereas deaths due to other causes were not different between the 2 groups. In addition, LV function of mice was assessed at 48 hours after banding; LV ejection fraction measured with echocardiography was lower (
P
<0.05) in the cyclosporine-treated banded group (66±3.0%) than in the nontreatment banded group (79±1.5%), whereas LV systolic wall stresses were similar. Calcineurin phosphatase activity was depressed similarly in both cyclosporine-treated groups compared with both nontreatment groups. Thus, cyclosporine could attenuate, but not prevent, LVH at the expense of inhibiting an important compensatory mechanism in response to pressure overload, resulting in reduced LV wall stress and function and increased susceptibility to decompensation and heart failure.
Correlation Between Myofilament Response to Ca
2+
and Altered Dynamics of Contraction and Relaxation in Transgenic Cardiac Cells That Express β-Tropomyosin
Authors: Wolska Beata M. , Keller Rebecca S. , Evans Christian C. , Palmiter Kimberly A. , Phillips Ronald M. , Muthuchamy Mariappan , Oehlenschlager James , Wieczorek David F. , de Tombe Pieter P. , Solaro R. John
Published: June 2012

Abstract
—We compared the dynamics of the contraction and relaxation of single myocytes isolated from nontransgenic (NTG) mouse hearts and from transgenic (TG-β-Tm) mouse hearts that overexpress the skeletal isoform of tropomyosin (Tm). Compared with NTG controls, TG-β-Tm myocytes showed significantly reduced maximal rates of contraction and relaxation with no change in the extent of shortening. This result indicated that the depression in contraction dynamics determined in TG-β-Tm isolated hearts is intrinsic to the cells. To further investigate the effect of Tm isoform switching on myofilament activity and regulation, we measured myofilament force and ATPase rate as functions of pCa (−log of [Ca
2+
]). Compared with controls, force generated by myofilaments from TG-β-Tm hearts and myofibrillar ATPase activity were both more sensitive to Ca
2+
. However, the shift in pCa
50
(half-maximally activating pCa) caused by changing sarcomere length from 1.8 to 2.4 μm was not significantly different between NTG and TG-β-Tm fiber preparations. To test directly whether isoform switching affected the economy of contraction, force versus ATPase rate relationships were measured in detergent-extracted fiber bundles. In both NTG and TG-β-Tm preparations, force and ATPase rate were linear and identically correlated, which indicated that crossbridge turnover was unaffected by Tm isoform switching. However, detergent extracted fibers from TG-β-Tm demonstrated significantly less maximum tension and ATPase activity than NTG controls. Our results provide the first evidence that the Tm isoform population modulates the dynamics of contraction and relaxation of single myocytes by a mechanism that does not alter the rate-limiting step of crossbridge detachment. Our results also indicate that differences in sarcomere-length dependence of activation between cardiac and skeletal muscle are not likely due to differences in the isoform population of Tm.
Thrombopoietin Stimulates Endothelial Cell Motility and Neoangiogenesis by a Platelet-Activating Factor–Dependent Mechanism
Authors: Brizzi Maria Felice , Battaglia Edda , Montrucchio Giuseppe , Dentelli Patrizia , Del Sorbo Lorenzo , Garbarino Giovanni , Pegoraro Luigi , Camussi Giovanni
Published: June 2012

Abstract
—In this study, we demonstrate that human umbilical cord vein–derived endothelial cells (HUVECs) expressed c-Mpl, the thrombopoietin (TPO) receptor, and that TPO activates HUVECs in vitro, as indicated by directional migration, synthesis of 1-alkyl-/1-acyl-platelet-activating factor (PAF) and interleukin-8 (IL-8), and phosphorylation of the signal transducers and activators of transcription (STAT) STAT1 and STAT5B. The observation that WEB 2170 and CV3988, 2 structurally unrelated PAF receptor antagonists, prevented the motility of HUVECs induced by TPO suggests a role of PAF as secondary mediator. Moreover, kinetic analysis of TPO-induced tyrosine phosphorylation of STAT demonstrated that STAT5B activation temporally correlated with the synthesis of PAF. PAF, in turn, induced a rapid tyrosine phosphorylation of STAT5B and PAF receptor blockade, by WEB 2170, preventing both TPO- and PAF-mediated STAT5B activation. The in vivo angiogenic effect of TPO, studied in a mouse model of Matrigel implantation, demonstrated that TPO induced a dose-dependent angiogenic response that required the presence of heparin. Moreover, the in vivo angiogenic effect of TPO was inhibited by the PAF receptor antagonist WEB 2170 but not by the anti–basic fibroblast growth factor neutralizing antibody. These results indicate that the effects of TPO are not restricted to cells of hematopoietic lineages, because TPO is able to activate endothelial cells and to induce an angiogenic response in which the recruitment of endothelial cells is mediated by the synthesis of PAF. Moreover, biochemical analysis supports the hypothesis that STAT5B may be involved in the signaling pathway leading to PAF-dependent angiogenesis.
Opioid-Induced Second Window of Cardioprotection
Authors: Fryer Ryan M. , Hsu Anna K. , Eells Janis T. , Nagase Hiroshi , Gross Garrett J.
Published: June 2012

Abstract
—Opioids have been previously shown to confer short-term cardioprotection against a prolonged ischemic insult. Therefore, the present study was designed to determine whether opioids can induce a delayed or “second window” of cardioprotection and to assess the potential involvement of the mitochondrial K
ATP
channel. All rats were subjected to 30 minutes of ischemia and 2 hours of reperfusion (I/R). Control animals, injected with saline 24 hours before I/R, elicited an infarct size/area at risk (IS/AAR) of 62.9±3.4. TAN-67, a δ1-opioid receptor agonist, was administered 10 or 30 mg/kg IP 12, 24, 48, or 72 hours before I/R. TAN-67 (10 mg/kg) 12- or 24-hour pretreatment did not significantly reduce IS/AAR (62.1±6.3 and 43.3±7.3, respectively). Similarly, 12-hour pretreatment with TAN-67 (30 mg/kg) did not reduce IS/AAR (60.0±5.6); however, 24-hour pretreatment significantly reduced IS/AAR (34.5±5.9). Forty-eight–hour pretreatment with TAN-67 maximally reduced IS/AAR (29.2±7.0), and opioid-induced cardioprotection was lost after 72-hour pretreatment (61.7±3.8). TAN-67–induced cardioprotection could be abolished by pretreatment with the selective δ
1
-opioid receptor antagonist 7-benzylidenenaltrexone, BNTX, administered either 30 minutes before TAN-67 given 48 hours before I/R or 10 minutes before I/R in rats previously treated for 48 hours with TAN-67 (59.6±3.1 and 58.7±3.5, respectively). The involvement of the K
ATP
channel was investigated with 2 inhibitors: glibenclamide, a nonselective K
ATP
channel inhibitor, and 5-hydroxydecanoic acid, selective for the mitochondrial K
ATP
channel in rabbits. Glibenclamide, administered 30 minutes before I/R in 48-hour TAN-67–pretreated rats, completely abolished cardioprotection (60.4±3.2). Similarly, 5-hydroxydecanoic acid, administered 5 minutes before I/R in rats pretreated 48 hours previously with TAN-67, completely abolished cardioprotection (57.8±2.5). These results suggest that δ
1
-opioid receptor stimulation, 24 to 48 hours before an ischemic insult, produces a delayed cardioprotective effect that is possibly the result of mitochondrial K
ATP
channel activation.