bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced Release of Prostaglandins Contributes to Flow-Induced Arteriolar Dilation in eNOS Knockout Mice
Authors: Sun Dong , Huang An , Smith Carolyn J. , Stackpole Christopher J. , Connetta Joseph A. , Shesely Edward G. , Koller Akos , Kaley Gabor
Published: June 2012

Abstract
—Nitric oxide and prostaglandins were shown to contribute to the endothelial mediation of flow-induced dilation of skeletal muscle arterioles of rats. Thus, we hypothesized that flow-induced dilation and its mediation are altered in gracilis muscle arterioles of mice deficient in the gene for endothelial nitric oxide synthase (eNOS-KO) compared with control wild-type (WT) mice. Gracilis muscle arterioles (≈80 μm) of male mice were isolated, then cannulated and pressurized in a vessel chamber. The increases in diameter elicited by increases in perfusate flow from 0 to 10 μL/min were similar in arterioles from eNOS-KO (n=28) and WT (n=22) mice (≈20 μm at 10 μL/min flow). Removal of the endothelium eliminated flow-induced dilations in vessels of both strains of mice.
N
ω
-nitro-
l
-arginine (L-NNA, 10
−4
mol/L) significantly inhibited flow-induced dilation in arterioles of WT mice by ≈51% but had no effect on responses of arterioles from eNOS-KO mice. Indomethacin (INDO, 10
−5
mol/L) inhibited flow-induced dilation of WT mice by ≈49%, whereas it completely abolished this response in arterioles of eNOS-KO mice. Simultaneous administration of INDO and L-NNA eliminated flow-induced responses in arterioles of WT mice. Dilations to carbaprostacyclin were similar at concentrations of 10
−8
and 3×10
−8
mol/L but decreased significantly at 10
−7
mol/L in arterioles of eNOS-KO compared with those of WT mice. These findings demonstrate that, despite the lack of nitric oxide mediation, flow-induced dilation is close to normal in arterioles of eNOS-KO mice because of an enhanced release of endothelial dilator prostaglandins and suggest that this vascular adaptation may contribute to the regulation of peripheral resistance in eNOS-KO mice.
A Role for Reactive Oxygen Species in Endothelial Cell Anoikis
Authors: Li Arthur E. , Ito Hideaki , Rovira Ilsa I. , Kim Kyung-Soo , Takeda Kazuyo , Yu Zu-Yi , Ferrans Victor J. , Finkel Toren
Published: June 2012

Abstract
—When adherent cells, such as epithelial or endothelial cells, are detached and continuously maintained in suspension, they undergo a form of programmed cell death termed anoikis. We demonstrate that coincident with endothelial cell detachment, there is a dramatic rise in the intracellular level of reactive oxygen species (ROS). Reattachment to a solid surface rapidly attenuates the level of ROS. The mitochondria appear to be the major source of the detachment-induced rise in ROS. The change in the intracellular redox state appears to contribute to endothelial anoikis, because treatment with either the cell-permeant antioxidant
N
-acetylcysteine or the flavin protein inhibitor diphenylene iodonium is demonstrated to reduce oxidant levels and protect against subsequent cell death. Similarly, the endogenous intracellular level of ROS is shown to correlate with the extent of cell death. Finally, we demonstrate that the activities of both caspases and of the c-Jun N-terminal kinases are modulated by the rise in intracellular ROS levels. These results suggest that oxidants serve as signaling molecules and regulators of anoikis.
E1A Can Provoke G1 Exit That Is Refractory to p21 and Independent of Activating Cdk2
Authors: Akli Saïd , Zhan Song , Abdellatif Maha , Schneider Michael D.
Published: June 2012

Abstract
—E1A can evoke G1 exit in cardiac myocytes and other cell types by displacing E2F transcription factors from tumor suppressor “pocket” proteins and by a less well-characterized p300-dependent pathway. Bypassing pocket proteins (through overexpression of E2F-1) reproduces the effect of inactivating pocket proteins (through E1A binding); however, pocket proteins associate with a number of molecular targets apart from E2F. Hence, pocket protein binding by E1A might engage mechanisms for cell cycle reentry beyond those induced by E2F-1. To test this hypothesis, we used adenoviral gene transfer to express various E2F-1 and E1A proteins in neonatal rat cardiac myocytes that are already refractory to mitogenic serum, in the absence or presence of several complementary cell cycle inhibitors—p16, p21, or dominant-negative cyclin-dependent kinase-2 (Cdk2). Rb binding by E2F-1 was neither necessary nor sufficient for G1 exit, whereas DNA binding was required; thus, exogenous E2F-1 did not merely function by competing for the Rb “pocket.” E2F-1–induced G1 exit was blocked by the “universal” Cdk inhibitor p21 but not by p16, a specific inhibitor of Cdk4/6; p21 was permissive for E2F-1 induction of cyclins E and A, but prevented their stimulation of Cdk2 kinase activity. In addition, E2F-1–induced G1 exit was blocked by dominant-negative Cdk2. Forced expression of cyclin E induced endogenous Cdk2 activity but not G1 exit. Thus, E2F-1–induced Cdk2 function was necessary, although not sufficient, to trigger DNA synthesis in cardiac muscle cells. In contrast, pocket protein–binding forms of E1A induced G1 exit that was resistant to inhibition by p21, whereas G1 exit via the E1A p300 pathway was sensitive to inhibition by p21. Both E1A pathways—via pocket proteins and via p300—upregulated cyclins E and A and Cdk2 activity, consistent with a role for Cdk2 in G1 exit induced by E1A. However, p21 blocked Cdk2 kinase activity induced by both E1A pathways equally. Thus, E1A can cause G1 exit without an increase in Cdk2 activity, if the pocket protein–binding domain is intact. E1A also overrides p21 in U2OS cells, provided the pocket protein–binding domain is intact; thus, this novel function of E1A is not exclusive to cardiac muscle cells. In summary, E1A binding to pocket proteins has effects beyond those produced by E2F-1 alone and can drive S-phase entry that is resistant to p21 and independent of an increase in Cdk2 function. This suggests the potential involvement of other endogenous Rb-binding proteins or of alternative E1A targets.
Mitochondrial Electron Transport Complex I Is a Potential Source of Oxygen Free Radicals in the Failing Myocardium
Authors: Ide Tomomi , Tsutsui Hiroyuki , Kinugawa Shintaro , Utsumi Hideo , Kang Dongchon , Hattori Nobutaka , Uchida Koji , Arimura Ken-ichi , Egashira Kensuke , Takeshita Akira
Published: June 2012

Abstract
—Oxidative stress in the myocardium may play an important role in the pathogenesis of congestive heart failure (HF). However, the cellular sources and mechanisms for the enhanced generation of reactive oxygen species (ROS) in the failing myocardium remain unknown. The amount of thiobarbituric acid reactive substances increased in the canine HF hearts subjected to rapid ventricular pacing for 4 weeks, and immunohistochemical staining of 4-hydroxy-2-nonenal ROS-induced lipid peroxides was detected in cardiac myocytes but not in interstitial cells of HF animals. The generation of superoxide anion was directly assessed in the submitochondrial fractions by use of electron spin resonance spectroscopy with spin trapping agent, 5,5′-dimethyl-1-pyrroline-
N
-oxide, in the presence of NADH and succinate as a substrate for NADH–ubiquinone oxidoreductase (complex I) and succinate–ubiquinone oxidoreductase (complex II), respectively. Superoxide production was increased 2.8-fold (
P
<0.01) in HF, which was due to the functional block of electron transport at complex I. The enzymatic activity of complex I decreased in HF (274±13 versus 136±9 nmol · min
−1
· mg
−1
protein,
P
<0.01), which may thus have caused the functional uncoupling of the respiratory chain and the deleterious ROS production in HF mitochondria. The present study provided direct evidence for the involvement of ROS in the mitochondrial origin of HF myocytes, which might be responsible for both contractile dysfunction and structural damage to the myocardium.
Atrial L-Type Ca
2+
Currents and Human Atrial Fibrillation
Authors: Van Wagoner David R. , Pond Amber L. , Lamorgese Michelle , Rossie Sandra S. , McCarthy Patrick M. , Nerbonne Jeanne M.
Published: June 2012

Abstract
—Chronic atrial fibrillation (AF) is characterized by decreased atrial contractility, shortened action potential duration, and decreased accommodation of action potential duration to changes in activation rate. Studies on experimental animal models of AF implicate a reduction in L-type Ca
2+
current (
I
Ca
) density in these changes. To evaluate the effect of AF on human
I
Ca
, we compared
I
Ca
in atrial myocytes isolated from 42 patients in normal sinus rhythm at the time of cardiac surgery with that of 11 chronic AF patients.
I
Ca
was significantly reduced in the myocytes of patients with chronic AF (mean −3.35±0.5 pA/pF versus −9.13±1.0 pA/pF in the controls), with no difference between groups in the voltage dependence of activation or steady-state inactivation. Although
I
Ca
was lower in myocytes from the chronic AF patients, their response to maximal β-adrenergic stimulation was not impaired. Postoperative AF frequently follows cardiac surgery. Half of the patients in the control group (19/38) of this study experienced postoperative AF. Whereas chronic AF is characterized by reduced atrial
I
Ca
, the patients with the greatest
I
Ca
had an increased incidence of postoperative AF, independent of patient age or diagnosis. This observation is consistent with the concept that calcium overload may be an important factor in the initiation of AF. The reduction in functional
I
Ca
density in myocytes from the atria of chronic AF patients may thus be an adaptive response to the arrhythmia-induced calcium overload.
Absence of Troponin I Degradation or Altered Sarcoplasmic Reticulum Uptake Protein Expression After Reversible Ischemia in Swine
Authors: Thomas Salome A. , Fallavollita James A. , Lee Te-Chung , Feng Jun , Canty John M.
Published: June 2012

Abstract
—The findings of troponin I (TnI) proteolysis (in isolated rat hearts) and induction of selected sarcoplasmic reticulum (SR) calcium-regulatory genes (after repetitive total coronary occlusions in swine) have given rise to the hypothesis that the time course of functional recovery of stunned myocardium reflects the resynthesis of reversibly damaged proteins. Although stunning occurs after brief total occlusions and prolonged partial occlusions (ie, short-term hibernation), the time course of functional recovery varies from a few hours to several days, suggesting that the severity of protein damage or mechanisms responsible for the dysfunction may differ. To study this, we examined SR gene expression and TnI degradation in stunned myocardium produced by 10-minute total left anterior descending coronary artery (LAD) occlusions (n=4) or 1-hour partial LAD occlusions, in which flow was reduced to ≈50% of control values for 60 minutes (n=6) in swine. One hour after reperfusion, LAD wall thickening was severely depressed in both models despite normal perfusion and no triphenyltetrazolium chloride evidence of necrosis. Normal myocardium exhibited TnI immunoreactivity at 31 kDa and a weak secondary band at 27 kDa. Irreversible injury or calpain activation in vitro produced a marked increase in the intensity of the 27-kDa band, consistent with TnI degradation. Stunned myocardium demonstrated no change in the 31- or the 27-kDa band, and the percentage of the 27- to 31-kDa band remained constant after 10-minute total occlusions (LAD, 5.9±0.9%; normal, 4.9±1.6%) and 1-hour partial occlusions (LAD, 8.5±1.9%; normal, 7.3±1.4%) and in sham controls (LAD, 10.9±1.5%; normal, 9.8±1.4%). Northern analysis showed no alterations in TnI or SR gene expression, but the stress protein HSP-70 was variably induced. Thus, stunned myocardium occurs without TnI degradation or altered SR gene expression, indicating that additional mechanisms are responsible for the reversible dysfunction after single episodes of regional ischemia in swine.
Thyroid Hormone Regulates Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel (HCN2) mRNA in the Rat Heart
Authors: Pachucki Janusz , Burmeister Lynn A. , Larsen P. Reed
Published: June 2012

Abstract
—Thyroid hormone regulation of the cardiac pacemaker gene, the hyperpolarization-activated cyclic nucleotide-gated channel gene (HCN2), was studied in rats by Northern analysis. Thyroid hormone administration to hypothyroid rats resulted in a doubling of the HCN2/β-actin mRNA ratio. A smaller, not statistically significant, increase in HCN2 mRNA occurred when euthyroid animals were made hyperthyroid. A single large dose of
l
-triiodothyronine given to hypothyroid rats caused a 4.7-fold increase in myocardial HCN2 mRNA expression level and only a 2.3-fold increase in the β-actin mRNA level. Although the rat HCN2 promoter has not been cloned, we identified a consensus thyroid hormone response element in the promoter sequence of the human HCN2 gene. Therefore, the increase in rat HCN2 mRNA is likely due to
l
-triiodothyronine stimulation of HCN2 gene transcription. The results suggest that the regulation of heart rate by thyroid hormone may be explained, at least in part, by the positive effect of this hormone on HCN2 gene expression.
Demonstration of Selective Protein Kinase C–Dependent Activation of Src and Lck Tyrosine Kinases During Ischemic Preconditioning in Conscious Rabbits
Authors: Ping Peipei , Zhang Jun , Zheng Yu-Ting , Li Richard C. X. , Dawn Buddhabeb , Tang Xian-Liang , Takano Hitoshi , Balafanova Zarema , Bolli Roberto
Published: June 2012

Abstract
—Src tyrosine kinases have been shown to mediate cellular responses to stress in noncardiac cells. However, the effect of myocardial ischemia on Src tyrosine kinases is unknown. Furthermore, the identity of the tyrosine kinase(s) involved in the genesis of ischemic preconditioning (PC) remains obscure. Here, we present the first evidence that ischemic PC (6 cycles of 4-minute coronary occlusion and 4-minute reperfusion) induces selective activation of 2 members of the Src family of tyrosine kinases, Src and Lck, in the heart of conscious rabbits. The activation of Src in the particulate fraction was not evident at 5 minutes after ischemic PC but became apparent at 30 minutes (+119% versus control), whereas the activation of Lck in the particulate fraction was apparent both at 5 minutes (+103% versus control) and at 30 minutes (+89%) after ischemic PC. The activity of the other 5 members of the Src tyrosine kinases expressed in the rabbit heart (Fyn, Fgr, Yes, Lyn, and Blk) was not affected by ischemic PC. Ischemic PC had no effect on the activity of epidermal growth factor receptor kinases, either at 5 or at 30 minutes. The activation of Src and Lck was completely abrogated by the tyrosine kinase inhibitor lavendustin A, given at doses that have previously been shown to block the protective effect of ischemic PC in this same conscious rabbit model, suggesting that Src and Lck kinases are essential for the development of ischemic PC. The activity of the ε isoform of protein kinase C (PKC) in the particulate fraction increased at 5 minutes (+72%) and at 30 minutes (+67%) after ischemic PC. Pretreatment with lavendustin A had no effect on the activation of PKCε, whereas pretreatment with the PKC inhibitor chelerythrine (given at doses that have previously been shown to block ischemic PC) blocked not only the activation of PKCε but also that of Src and Lck, indicating that Src and Lck are downstream of PKCε in the signaling cascade of ischemic PC. This study identifies a new component of the signaling mechanism of ischemic PC. The results support the concept that, in conscious rabbits, 2 specific members of the Src family of tyrosine kinases, Src and Lck, play an important role in the genesis of late PC by serving as downstream elements of PKC-mediated signal transduction.
Predominant Distribution of Nifedipine-Insensitive, High Voltage–Activated Ca
2+
Channels in the Terminal Mesenteric Artery of Guinea Pig
Authors: Morita Hiromitsu , Cousins Helen , Onoue Hitoshi , Ito Yushi , Inoue Ryuji
Published: June 2012

Abstract
—We have found nifedipine-insensitive (NI), rapidly inactivating, voltage-dependent Ca
2+
channels (current, NI-
I
Ca
) with unique biophysical and pharmacological properties in the terminal branches of guinea pig mesenteric artery, by using a whole-cell mode of the patch-clamp technique. The fraction of NI-
I
Ca
appeared to increase dramatically along the lower branches of mesenteric artery, amounting to almost 100% of global
I
Ca
in its periphery. With 5 mmol/L Ba
2+
as the charge carrier, NI-
I
Ca
was activated with a threshold of −50 mV, peaked at −10 mV, and was half-activated and inactivated at −11 and −52 mV, respectively, generating a potential range of constant activation near the resting membrane potential. The NI-
I
Ca
was rundown resistant, was not subject to Ca
2+
-dependent inactivation, and exhibited the pore properties typical for high voltage–activated Ca
2+
channels; Ba
2+
is ≈2-fold more permeable than Ca
2+
, and Cd
2+
is a better blocker than Ni
2+
(IC
50
, 6 and 68 μmol/L, respectively). Relatively specific blockers for N- and P/Q-type Ca
2+
channels such as ω-conotoxins GVIA and MVIIC (each 1 μmol/L) and ω-agatoxin IVA (1 μmol/L) were ineffective at inhibiting NI-
I
Ca
, whereas nimodipine partially (10 μmol/L; ≈40%) and amiloride potently (≈75% with 1 mmol/L; IC
50
; 107 μmol/L) blocked the current. Although these properties are reminiscent of R-type Ca
2+
channels, expression of the α
1E
mRNA was not detected using reverse transcriptase–polymerase chain reaction. These results strongly suggest the predominant presence of NI, high voltage–activated Ca
2+
channels with novel properties, which may be abundantly expressed in peripheral small arterioles and contribute to their tone regulation.
Adenosine Receptor Activation Induces Vascular Endothelial Growth Factor in Human Retinal Endothelial Cells
Authors: Grant Maria B. , Tarnuzzer Roy W. , Caballero Sergio , Ozeck Mark J. , Davis Margaret I. , Spoerri Polyxenie E. , Feoktistov Igor , Biaggioni Italo , Shryock John C. , Belardinelli Luiz
Published: June 2012

Abstract
—Adenosine, released in increased amounts by hypoxic tissues, is thought to be an angiogenic factor that links altered cellular metabolism caused by oxygen deprivation to compensatory angiogenesis. Adenosine interacts with 4 subtypes of G protein–coupled receptors, termed A
1
, A
2A
, A
2B
, and A
3
. We investigated whether adenosine causes proliferation of human retinal endothelial cells (HRECs) and synthesis of vascular endothelial growth factor (VEGF) and, if so, which adenosine receptor subtype mediates these effects. The nonselective adenosine receptor agonist 5′-
N
-ethylcarboxamidoadenosine (NECA), in a concentration-dependent manner, increased both VEGF mRNA and protein expression by HRECs, as well as proliferation. This proliferative effect of NECA was inhibited by the addition of anti-human VEGF antibody. NECA also increased insulin-like growth factor-I and basic fibroblast growth factor mRNA expression in a time-dependent manner and cAMP accumulation in these cells. In contrast, neither the A
1
agonist
N
6
-cyclopentyladenosine nor the A
2A
agonist 2-
p
-(2-carboxyethyl) phenethylamino-NECA caused any of the above effects of NECA. The effects of NECA were not significantly attenuated by either the A
2A
antagonist
SCH58261
or the A
1
antagonist 8-cyclopentyl-1,3-dipropylxanthine. However, the nonselective adenosine receptor antagonist xanthine amine congener completely inhibited the effects of NECA. Addition of antisense oligonucleotide complementary to A
2B
adenosine receptor mRNA inhibited VEGF protein production by HRECs after NECA stimulation. Thus, the A
2B
adenosine receptor subtype appears to mediate the actions of adenosine to increase growth factor production, cAMP content, and cell proliferation of HRECs. Adenosine activates the A
2B
adenosine receptor in HRECs, which may lead to neovascularization by a mechanism involving increased angiogenic growth factor expression.
A Flow-Activated Chloride-Selective Membrane Current in Vascular Endothelial Cells
Authors: Barakat A. I. , Leaver E. V. , Pappone P. A. , Davies P. F.
Published: June 2012

Abstract
—Shear stress–induced activation of endothelial ion channels, one of the earliest responses to flow, is implicated in mechano–signal transduction that results in the regulation of vascular tone. The effects of laminar flow on endothelial membrane potential were studied in vitro using both fluorescent potentiometric dye measurements and whole-cell patch-clamp recordings. The application of flow stimulated membrane hyperpolarization, which was reversed to depolarization within 35 to 160 seconds. The depolarization was caused by a Cl
−
-selective membrane current activated by flow independently of the K
+
channel-mediated hyperpolarization. Thus, flow activated both K
+
and Cl
−
currents, with the net membrane potential being determined by the balance of the responses. Membrane potential sensitivity to flow was unchanged by flow preconditioning that elongated and aligned the cells.
Cytokine-Mediated Apoptosis in Cardiac Myocytes
Authors: Arstall Margaret A. , Sawyer Douglas B. , Fukazawa Ryuji , Kelly Ralph A.
Published: June 2012

Abstract
—Increased production of nitric oxide (NO) after induction of the cytokine-inducible isoform of nitric oxide synthase (iNOS or NOS2) in cardiac myocytes and other parenchymal cells within the heart may in addition to contributing to myocyte contractile dysfunction also contribute to the induction of programmed cell death (apoptosis). To investigate the mechanism(s) by which increased NO production leads to apoptosis, we examined the role of NO in primary cultures of neonatal rat ventricular myocytes (NRVMs) after induction by the cytokines interleukin-1β (IL-1β) and interferon γ (IFNγ) or exposure to the exogenous NO donor
S
-nitroso-
N
-acetylcysteine (SNAC) or peroxynitrite (ONOO
−
). Both SNAC (1 mmol/L) and ONOO
−
(100 μmol/L), but not their respective controls (ie,
N
-acetylcysteine and pH-inactivated ONOO
−
), induced apoptosis in confluent, serum-starved NRVMs at 48 hours. Similarly, incubation of NRVMs with IL-1β and IFNγ for 48 hours resulted in an increase in iNOS expression, nitrite production, and programmed cell death. Both the cytokine-induced nitrite accumulation and myocyte apoptosis could be completely prevented by the nonselective NOS inhibitor
l
-nitroarginine (3 mmol/L) or the specific iNOS inhibitor 2-amino-5,6-dihydro-6-methyl-4
H
-1,3-thiazine (AMT, 100 μmol/L). NO-mediated myocyte apoptosis was not attenuated by the inhibition of soluble guanylyl cyclase with ODQ, nor could apoptosis be induced by the incubation of NRVMs with 1 mmol/L 8-bromo-cGMP, a cell-permeant cGMP analogue. However, NO-mediated apoptosis was significantly attenuated by the superoxide dismutase mimetic and ONOO
−
scavenger Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP, 100 μmol/L). NO/ONOO
−
-mediated apoptosis was associated with increased expression of Bax with no change in Bcl-2 mRNA abundance. Furthermore, apoptotic cell death was also confirmed in adult rat ventricular myocytes (ARVMs) when grown in heteroculture with IL-1β– and IFNγ-treated rat cardiac microvascular endothelial cells. Therefore, cytokine-induced apoptosis in NRVMs and ARVMs is mediated by iNOS induction, ONOO
−
, and associated with an increase in Bax levels.
Myocyte Death in the Failing Human Heart Is Gender Dependent
Authors: Guerra Sabrina , Leri Annarosa , Wang Xiaowei , Finato Nicoletta , Di Loreto Carla , Beltrami Carlo Alberto , Kajstura Jan , Anversa Piero
Published: June 2012

Abstract
—Cardiovascular disease is delayed and less common in women than in men. Myocyte death occurs in heart failure, but only apoptosis has been documented; the role of myocyte necrosis is unknown. Therefore, we tested whether necrosis is as important as apoptosis and whether myocyte death is lower in women than in men with heart failure. Molecular probes were used to measure the magnitude of myocyte necrosis and apoptosis in 7 women and 12 men undergoing transplantation for cardiac failure. Myocyte necrosis was evaluated by detection of DNA damage with blunt end fragments, whereas apoptosis was assessed by the identification of double-strand DNA cleavage with single base or longer 3′ overhangs. An identical analysis of these forms of cell death was performed in control myocardium. Heart failure showed levels of myocyte necrosis 7-fold greater than apoptosis in patients of both sexes. However, cell death was 2-fold higher in men than in women. Heart failure resulted in a 13-fold and 27-fold increase in necrosis in women and men, respectively. Apoptosis increased 35-fold in women and 85-fold in men. The differences in cell death between women and men were confirmed by the electrophoretic pattern of DNA diffusion and laddering of isolated myocytes. The lower degree of cell death in women was associated with a longer duration of the myopathy, a later onset of cardiac decompensation, and a longer interval between heart failure and transplantation. In conclusion, myocyte necrosis and apoptosis affect the decompensated human heart; each contributes to the evolution of cardiac failure. However, the female heart is protected, at least in part, from necrotic and apoptotic death signals.
Angiopoietin-1 Regulates Endothelial Cell Survival Through the Phosphatidylinositol 3′-Kinase/Akt Signal Transduction Pathway
Authors: Kim Injune , Kim Hwan Gyu , So June-No , Kim Joo Heon , Kwak Hee Jin , Koh Gou Young
Published: June 2012

Abstract
—Angiopoietin-1 (Ang1) is a strong apoptosis survival factor for endothelial cells. In this study, the receptor/second messenger signal transduction pathway for the antiapoptotic effect of Ang1 on human umbilical vein endothelial cells was examined. Pretreatment with soluble Tie2 receptor, but not Tie1 receptor, blocked the Ang1-induced antiapoptotic effect. Ang1 induced phosphorylation of Tie2 and the p85 subunit of phosphatidylinositol 3′-kinase (PI 3′-kinase) and increased PI 3′-kinase activity in a dose-dependent manner. The PI 3′-kinase–specific inhibitors wortmannin and LY294002 blocked the Ang1-induced antiapoptotic effect. Ang1 induced phosphorylation of the serine-threonine kinase Akt at Ser473 in a PI 3′-kinase–dependent manner. Expression of a dominant-negative form of Akt reversed the Ang1-induced antiapoptotic effect. Ang1 mRNA and protein were present in vascular smooth muscle cells but not in endothelial cells. Cultured vascular smooth muscle cells, but not human umbilical vein endothelial cells, secreted Ang1. These findings indicate that the Tie2 receptor, PI 3′-kinase, and Akt are crucial elements in the signal transduction pathway leading to endothelial cell survival induced by the paracrine activity of Ang1.
Mechanism of Doxorubicin-Induced Inhibition of Sarcoplasmic Reticulum Ca
2+
-ATPase Gene Transcription
Authors: Arai Masashi , Yoguchi Akemi , Takizawa Takako , Yokoyama Tomoyuki , Kanda Tsugiyasu , Kurabayashi Masahiko , Nagai Ryozo
Published: June 2012

Abstract
—Doxorubicin (DOX)–induced cardiomyopathy has been found to be associated with impaired Ca
2+
handling in the sarcoplasmic reticulum (SR), leading to reduced cardiac function. We have recently demonstrated that expression of mRNA encoding sarco(endo)plasmic reticulum Ca
2+
-ATPase 2 (SERCA2), a major Ca
2+
transport protein in SR, is markedly decreased in DOX-treated hearts. To extend this observation, we have dissected the molecular mechanisms by which DOX downregulates SERCA2 gene transcription. Using cultured rat neonatal cardiac myocytes, we found that the antioxidant
N
-acetylcysteine blocked the DOX-induced decrease in SERCA2 mRNA levels, as well as the DOX-induced increase in H
2
O
2
concentration; thus, H
2
O
2
is an intracellular mediator of DOX activity. Using a luciferase reporter assay, we found that the sequence from −284 to −72 bp in the 5′ flanking region of the SERCA2 gene has a DOX-responsive element. Although several transcription factors have putative binding motifs in this region of the SERCA2 gene, only the expression of Egr-1 mRNA and the binding of Egr-1 protein to the 5′ regulatory sequence of SERCA2 gene increased markedly after DOX administration. We also found that overexpression of Egr-1 was associated with a significant reduction in SERCA2 gene transcription. In addition, Egr-1 antisense oligonucleotides blocked the DOX-induced reduction in SERCA2 mRNA, suggesting that Egr-1 is a transcriptional inhibitor of the SERCA2 gene in DOX-induced cardiomyopathy. We observed activation of 3 mitogen-activated protein kinases (MAPKs), p44/42 MAPK, p38 MAPK, and stress-activated MAPK/Jun N-terminal kinase, by DOX, but only a specific inhibitor of the p44/42 MAPK kinase suppressed the effects of DOX on Egr-1 and SERCA2 mRNA expression. These findings indicate that reactive oxygen intermediates, the transcription factor Egr-1, and p44/42 MAPK are critical elements in the transcriptional regulation of the SERCA2 gene in response to DOX.
Vascular Endothelial Growth Factor
165
Gene Transfer Augments Circulating Endothelial Progenitor Cells in Human Subjects
Authors: Kalka Christoph , Masuda Haruchika , Takahashi Tomono , Gordon Rebecca , Tepper Oren , Gravereaux Edwin , Pieczek Ann , Iwaguro Hideki , Hayashi Shin-Ichiro , Isner Jeffrey M. , Asahara Takayuki
Published: June 2012

Abstract
—Preclinical studies in animal models and early results of clinical trials in patients suggest that intramuscular injection of naked plasmid DNA encoding vascular endothelial growth factor (VEGF) can promote neovascularization of ischemic tissues. Such neovascularization has been attributed exclusively to sprout formation of endothelial cells derived from preexisting vessels. We investigated the hypothesis that VEGF gene transfer may also augment the population of circulating endothelial progenitor cells (EPCs). In patients with critical limb ischemia receiving VEGF gene transfer, gene expression was documented by a transient increase in plasma levels of VEGF. A culture assay documented a significant increase in EPCs (219%,
P
<0.001), whereas patients who received an empty vector had no change in circulating EPCs, as was the case for volunteers who received saline injections (VEGF versus empty vector,
P
<0.001; VEGF versus saline,
P
<0.005). Fluorescence-activated cell sorter analysis disclosed an overall increase of up to 30-fold in endothelial lineage markers KDR (VEGF receptor-2), VE-cadherin, CD34, α
v
β
3
, and E-selectin after VEGF gene transfer. Constitutive overexpression of VEGF in patients with limb ischemia augments the population of circulating EPCs. These findings support the notion that neovascularization of human ischemic tissues after angiogenic growth factor therapy is not limited to angiogenesis but involves circulating endothelial precursors that may home to ischemic foci and differentiate in situ through a process of vasculogenesis.
Postanoxic T Lymphocyte–Endothelial Cell Interactions Induce Tumor Necrosis Factor-α Production and Neutrophil Adhesion
Authors: Kokura Satoshi , Wolf Robert E. , Yoshikawa Toshikazu , Granger D. Neil , Aw Tak Yee
Published: June 2012

Abstract
—The objective of this study was to define the influence of postanoxic T-lymphocyte–endothelial cell interactions on anoxia-reoxygenation (A/R)–induced neutrophil–endothelial cell adhesion and cell adhesion molecule (CAM) expression on human umbilical vein endothelial cells (HUVECs). HUVEC monolayers were exposed to 60 minutes of anoxia, followed by 24 hours of reoxygenation, wherein freshly isolated human T lymphocytes were added at 6 hours during reoxygenation. After an additional 18 hours of incubation (ie, total of 24 hours of reoxygenation), the T-cell/endothelial cell (TC/EC) coculture media were collected and added to naive HUVEC monolayers incubated with neutrophils. Although the A/R-conditioned media per se had no effect on neutrophil adhesion, the media from TC/EC cocultures significantly increased the adhesion response. This enhanced adhesive interaction was associated with significant increases in tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) levels in the TC/EC coculture media and was accompanied by a pronounced increase in endothelial E-selectin expression. Treatment of the TC/EC coculture media with anti–TNF-α or anti-IL-8 antibodies reduced the media-induced neutrophil adhesion response. The enhanced neutrophil adhesion and the elevated medium levels of TNF-α, but not IL-8, were markedly reduced by inserts that prevented direct TC/EC contact and by monoclonal antibodies directed against vascular cell adhesion molecule-1 (VCAM-1) or very late antigen-4 (VLA-4). Collectively, these findings show that VLA-4–/VCAM-1–mediated interactions between T lymphocytes and postanoxic endothelial cells stimulates TNF-α production, which in turn elicits endothelial cell adhesion molecule expression and a corresponding increase in neutrophil adhesion.
Regulation of Protein Kinase B and 4E-BP1 by Oxidative Stress in Cardiac Myocytes
Authors: Pham Fong H. , Sugden Peter H. , Clerk Angela
Published: June 2012

Abstract
—Stimulation of phosphatidylinositol 3′-kinase (PI3K) and protein kinase B (PKB) is implicated in the regulation of protein synthesis in various cells. One mechanism involves PI3K/PKB-dependent phosphorylation of 4E-BP1, which dissociates from eIF4E, allowing initiation of translation from the 7-methylGTP cap of mRNAs. We examined the effects of insulin and H
2
O
2
on this pathway in neonatal cardiac myocytes. Cardiac myocyte protein synthesis was increased by insulin, but was inhibited by H
2
O
2
. PI3K inhibitors attenuated basal levels of protein synthesis and inhibited the insulin-induced increase in protein synthesis. Insulin or H
2
O
2
increased the phosphorylation (activation) of PKB through PI3K, but, whereas insulin induced a sustained response, the response to H
2
O
2
was transient. 4E-BP1 was phosphorylated in unstimulated cells, and 4E-BP1 phosphorylation was increased by insulin. H
2
O
2
stimulated dephosphorylation of 4E-BP1 by increasing protein phosphatase (PP1/PP2A) activity. This increased the association of 4E-BP1 with eIF4E, consistent with H
2
O
2
inhibition of protein synthesis. The effects of H
2
O
2
were sufficient to override the stimulation of protein synthesis and 4E-BP1 phosphorylation induced by insulin. These results indicate that PI3K and PKB are important regulators of protein synthesis in cardiac myocytes, but other factors, including phosphatase activity, modulate the overall response.
Direct Evidence for Increased Hydroxyl Radicals Originating From Superoxide in the Failing Myocardium
Authors: Ide Tomomi , Tsutsui Hiroyuki , Kinugawa Shintaro , Suematsu Nobuhiro , Hayashidani Shunji , Ichikawa Kazuhiro , Utsumi Hideo , Machida Youji , Egashira Kensuke , Takeshita Akira
Published: June 2012

Abstract
—Experimental and clinical studies have suggested an increased production of reactive oxygen species (ROS) in the failing myocardium. The present study aimed to obtain direct evidence for increased ROS and to determine the contribution of superoxide anion (·O
2
−
), H
2
O
2
, and hydroxy radical (·OH) in failing myocardial tissue. Heart failure was produced in adult mongrel dogs by rapid ventricular pacing at 240 bpm for 4 weeks. To assess the production of ROS directly, freeze-clamped myocardial tissue homogenates were reacted with the nitroxide radical, 4-hydroxy-2,2,6,6,-tetramethyl-piperidine-
N
-oxyl, and its spin signals were detected by electron spin resonance spectroscopy. The rate of electron spin resonance signal decay, proportional to ·OH level, was significantly increased in heart failure, which was inhibited by the addition of dimethylthiourea (·OH scavenger) into the reaction mixture. Increased ·OH in the failing heart was abolished to the same extent in the presence of desferrioxamine (iron chelator), catalase (H
2
O
2
scavenger), and 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron; LaMotte) (·O
2
−
scavenger), indicating that ·OH originated from H
2
O
2
and ·O
2
−
. Further, ·O
2
−
produced in normal myocardium in the presence of antimycin A (mitochondrial complex III inhibitor) could reproduce the increase of H
2
O
2
and ·OH seen in the failing tissue. There was a significant positive relation between myocardial ROS level and left ventricular contractile dysfunction. In conclusion, in the failing myocardium, ·OH was produced as a reactive product of ·O
2
−
and H
2
O
2
, which might play an important role in left ventricular failure.
Rapid Activation of Neutral Sphingomyelinase by Hypoxia-Reoxygenation of Cardiac Myocytes
Authors: Hernandez Olga M. , Discher Daryl J. , Bishopric Nanette H. , Webster Keith A.
Published: June 2012

Abstract
—Elevated levels of oxygen free radicals have been implicated in the pathways of reperfusion injury to myocardial tissue. The targets for free radicals may include specific as well as random intracellular components, and part of the cellular response is the induction of extracellularly activated and stress-activated kinases. The intermediate signals that initiate these stress responses are not known. Here we show that one of the earliest responses of cardiac myocytes to hypoxia and reoxygenation is the activation of neutral sphingomyelinase and accumulation of ceramide. Ceramide increased abruptly after reoxygenation, peaking at 10 minutes with 225±40% of the control level. Neutral sphingomyelinase activity was induced with similar kinetics, and both activities remained elevated for several hours. c-Jun N-terminal kinase (JNK) was also activated within the same time frame. Treatment of cardiac myocytes with extracellular ceramides also activated JNK. Pretreating cells with antioxidants quenched sphingomyelinase activation, ceramide accumulation, and JNK activation. Ceramide did not accumulate in reoxygenated nonmuscle fibroblasts, and JNK was not activated by reoxygenation in these cells. The results identify neutral sphingomyelinase activation as one of the earliest responses of cardiac myocytes to the redox stress imposed by hypoxia-reoxygenation. The results are consistent with a pathway of ceramide-mediated activation of JNK.
Intussusceptive Angiogenesis
Authors: Djonov V. , Schmid M. , Tschanz S. A. , Burri P. H.
Published: June 2012

Abstract
—Intussusceptive angiogenesis is a novel mode of blood vessel formation and remodeling, which occurs by internal division of the preexisting capillary plexus without sprouting. In this study, the process is demonstrated in developing chicken eye vasculature and in the chorioallantoic membrane by methylmethacrylate (Mercox) casting, transmission electron microscopy, and in vivo observation. In a first step of intussusceptive angiogenesis, the capillary plexus expands by insertion of numerous transcapillary tissue pillars, ie, by intussusceptive microvascular growth. In a subsequent step, a vascular tree arises from the primitive capillary plexus as a result of intussusceptive pillar formation and pillar fusions, a process we termed “intussusceptive arborization.” On the basis of the morphological observations, a 4-step model for intussusceptive arborization is proposed, as follows: phase I, numerous circular pillars are formed in rows, thus demarcating future vessels; phase II, formation of narrow tissue septa by pillar reshaping and pillar fusions; phase III, delineation, segregation, growth, and extraction of the new vascular entity by merging of septa; and phase IV, formation of new branching generations by successively repeating the process, complemented by growth and maturation of all components. In contrast to sprouting, intussusceptive angiogenesis does not require intense local endothelial cell proliferation; it is implemented primarily by rearrangement and attenuation of the endothelial cell plates. In summary, transcapillary pillar formation, ie, intussusception, is a central and probably widespread process, which plays a role not only in capillary network growth and expansion (intussusceptive microvascular growth), but also in vascular plexus remodeling and tree formation (intussusceptive arborization).
Transcriptional and Posttranscriptional Regulation of Endothelial Nitric Oxide Synthase Expression by Hydrogen Peroxide
Authors: Drummond Grant R. , Cai Hua , Davis Michael E. , Ramasamy Santhini , Harrison David G.
Published: June 2012

Abstract
—Diverse stimuli, including shear stress, cyclic strain, oxidized LDL, hyperglycemia, and cell growth, modulate endothelial nitric oxide synthase (eNOS) expression. Although seemingly unrelated, these may all alter cellular redox state, suggesting that reactive oxygen intermediates might modulate eNOS expression. The present study was designed to test this hypothesis. Exposure of bovine aortic endothelial cells for 24 hours to paraquat, a superoxide (O
2
−·
)–generating compound, did not affect eNOS mRNA levels. However, cotreatment with paraquat and either Cu
2+
/Zn
2+
superoxide dismutase or the superoxide dismutase mimetic tetrakis(4-benzoic acid)porphyrin chloride increased eNOS mRNA by 2.3- and 2.2-fold, respectively, implicating a role for H
2
O
2
. Direct addition of 100 and 150 μmol/L H
2
O
2
caused increases in bovine aortic endothelial cell eNOS mRNA that were dependent on concentration (ie, 3.1- and 5.2-fold increases) and time, and elevated eNOS protein expression and enzyme activity, accordingly. Nuclear run-on and 5,6-dichloro-1-β-
d
-ribofuranosylbenzimidazole–chase studies showed that H
2
O
2
caused a 3.0-fold increase in eNOS gene transcription and a 2.8-fold increase in eNOS mRNA half-life. Induction of eNOS by H
2
O
2
was not affected by the hydroxyl radical scavenger DMSO, mannitol, or
N-tert-butyl
-α-phenylnitrone, but it was inhibited by the antioxidants
N
-acetylcysteine, ebselen, and exogenously added catalase. Unlike H
2
O
2
, the 4.0-fold induction of eNOS by shear stress (15 dyne/cm
2
for 6 hours) was not inhibited by
N
-acetylcysteine or exogenous catalase. In conclusion, H
2
O
2
increases eNOS expression through transcriptional and post-transcriptional mechanisms. Although H
2
O
2
does not mediate shear-dependent eNOS regulation, it is likely to be involved in regulation of eNOS expression in response to other physiological and/or pathophysiological stimuli.
Kallistatin Stimulates Vascular Smooth Muscle Cell Proliferation and Migration In Vitro and Neointima Formation in Balloon-Injured Rat Artery
Authors: Miao Robert Q. , Murakami Hideyuki , Song Qing , Chao Lee , Chao Julie
Published: June 2012

Abstract
—Kallistatin, a serine proteinase inhibitor (serpin), is expressed in the endothelial and smooth muscle cells of blood vessels. The potential function of kallistatin in vascular biology was investigated by studying its role in the proliferation and migration of cultured primary aortic vascular smooth muscle cells (VSMCs) in vitro and in neointima formation in rat artery after balloon angioplasty in vivo. Exogenous kallistatin induced a >2-fold increase of VSMC proliferation and cell growth as measured by [
3
H]thymidine incorporation and cell counts and a 2.3-fold increase of cell migration in modified Boyden chambers. In balloon-injured vessels, endogenous kallistatin mRNA and protein levels increased up to 10-fold as determined by competitive polymerase chain reaction and by ELISA. Intense staining of kallistatin mRNA was identified in the proliferating VSMCs of balloon-injured arteries during cell migration from media to neointima by in situ hybridization histochemistry and immunohistochemistry. We observed an induction of kallistatin expression by platelet-derived growth factor (PDGF) and upregulation of p42/44 mitogen-activated protein kinase (MAPK) activity by kallistatin in cultured VSMCs. Conversely, adenovirus-mediated transfer of kallistatin antisense cDNA into cultured VSMCs inhibited PDGF-induced p42/44 MAPK activity and cell proliferation. Furthermore, local delivery of adenovirus carrying kallistatin antisense cDNA significantly downregulated kallistatin mRNA levels and attenuated neointima formation in balloon-injured rat arteries in vivo. These results indicate that kallistatin may play an important role in mediating PDGF-induced MAPK pathway on VSMC proliferation and in neointima formation after balloon angioplasty.
Coronary Microvascular Endothelial Cell Redox State in Left Ventricular Hypertrophy
Authors: Lang Derek , Mosfer Salah I. , Shakesby Alison , Donaldson Francis , Lewis Malcolm J.
Published: June 2012

Abstract
—Left ventricular hypertrophy (LVH) is associated with elevated plasma angiotensin II (Ang II) levels and endothelial dysfunction. The relationship between Ang II and endothelial dysfunction remains unknown, however, but it may involve an alteration in endothelial cell redox state. We therefore investigated the effect of Ang II on NADH/NADPH oxidase–mediated superoxide anion (O
2
−
) production by cultured guinea pig coronary microvascular endothelial cells (CMVEs) and CMVEs freshly isolated from a guinea pig, pressure-overload model of LVH. Lucigenin chemiluminescence was used to measure O
2
−
production in the particulate fraction of CMVE lysates. In cultured cells, incubation with Ang II (0.1 nmol/L to 1 μmol/L for 18 hours) resulted in significant (
P
<0.01) increases in both NADH- and NADPH-dependent O
2
−
production, with a peak effect at 1 nmol/L. The latter was significantly (
P
<0.01) inhibited by the AT
1
receptor antagonist losartan (1 μmol/L for 18 hours). In contrast, the O
2
−
response to Ang II (0.1 nmol/L to 1 μmol/L for 18 hours) was largely unaffected by concomitant exposure to the AT
2
antagonist PD 123319 (1 μmol/L). In freshly isolated CMVEs from nonoperated animals, NADH- and NADPH-dependent O
2
−
production was not different from that in sham-operated animals but was significantly (
P
<0.05) elevated in the aortic-banded animals. Plasma Ang II levels were significantly (
P
<0.001) elevated in the aortic-banded (1.25±0.12 μg/L, n=12) compared with sham-operated animals (0.63±0.06 μg/L, n=12). These data suggest that the endothelial dysfunction associated with LVH may be due, at least in part, to the Ang II–induced upregulation of NADH/NADPH oxidase-dependent O
2
−
production.
A Novel Anionic Inward Rectifier in Native Cardiac Myocytes
Authors: Duan Dayue , Ye Lingyu , Britton Fiona , Horowitz Burton , Hume Joseph R.
Published: June 2012

Abstract
—Although the cationic inward rectifiers (Kir and hyperpolarization-activated
I
f
channels) have been well characterized in cardiac myocytes, the expression and physiological role of anionic inward rectifiers in heart are unknown. In the present study, we report the functional and molecular identification of a novel chloride (Cl
−
) inward rectifier (Cl.ir) in mammalian heart. Under conditions in which cationic inward rectifier channels were blocked, membrane hyperpolarization (−40 to −140 mV) activated an inwardly rectifying whole-cell current in mouse atrial and ventricular myocytes. Under isotonic conditions, the current activated slowly with a biexponential time course (time constants averaging 179.7±23.4 [mean±SEM] and 2073.6±287.6 ms at −120 mV). Hypotonic cell swelling accelerated the activation and increased the current amplitude whereas hypertonic cell shrinkage inhibited the current. The inwardly rectifying current was carried by Cl
−
(
I
Cl.ir
) and had an anion permeability sequence of Cl
−
>
I
−
≫aspartate.
I
Cl.ir
was blocked by 9-anthracene-carboxylic acid and cadmium but not by stilbene disulfonates and tamoxifen. A similar
I
Cl.ir
was also observed in guinea pig cardiac myocytes. The properties of
I
Cl.ir
are consistent with currents generated by expression of ClC-2 Cl
−
channels. Reverse transcription polymerase chain reaction and Northern blot analysis confirmed transcriptional expression of ClC-2 in both atrial and ventricular tissues and isolated myocytes of mouse and guinea pig hearts. These results indicate that a novel
I
Cl.ir
is present in mammalian heart and support a potentially important role of ClC-2 channels in the regulation of cardiac electrical activity and cell volume under physiological and pathological conditions. The full text of this article is available at http://www.circresaha.org.