bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Mitochondrial [NADH] by Cytosolic [Ca
2+
] and Work in Trabeculae From Hypertrophic and Normal Rat Hearts
Authors: Brandes Rolf , Maier Lars S. , Bers Donald M.
Published: June 2012

Abstract
—Pressure overload hypertrophy has previously been shown to reduce contractility but paradoxically to increase O
2
consumption rates at a given force. Because O
2
consumption rates are related to mitochondrial [NADH] ([NADH]
m
), we tested the hypothesis that with hypertrophy, control of [NADH]
m
may be altered. Left ventricular trabeculae were isolated from banded and control rat hearts, and fluorescence spectroscopy was used to monitor [NADH]
m
and cytosolic [Ca
2+
] ([Ca
2+
]
c
). The hearts from banded rats developed hypertrophy (heart-to-body weight ratio increased from 4.1±0.1 to 4.9±0.1 mg/g) and hypertension (systolic arterial pressure increased from 117±4 to 175±5 mm Hg). Muscle workload was increased by stepwise increases in pacing frequency (up to 2 Hz). After increased work, [NADH]
m
fell and then slowly recovered toward control levels. When work was decreased, [NADH]
m
overshot control values and then slowly returned. The Ca
2+
-independent initial fall was larger for trabeculae from rats with hypertrophied hearts than from control rats (eg, 17±2% versus 11±1% when work was increased by increasing the frequency from 0.25 to 1 Hz). At 1 Hz, average [Ca
2+
]
c
was ≈280 nmol/L, and the Ca
2+
-dependent [NADH]
m
recovery was larger for trabeculae from rats with hypertrophied hearts (17±4% versus 10±2%) despite similar average [Ca
2+
]
c
. At steady state after Ca
2+
-dependent recovery, there was no difference in [NADH]
m
(fall of 1±2% versus 1±1%). Furthermore, the Ca
2+
-dependent overshoot was larger for trabeculae from hypertrophied than from control hearts (increase of 14±2% versus 9±2% when frequency was decreased from 1 to 0.25 Hz). We conclude that (1) there is initially a larger imbalance in NADH production versus consumption rate in hypertrophy (because NADH fell more) and (2) the Ca
2+
-dependent recovery mechanism is enhanced in hypertrophy (because NADH recovered and overshot more), thus compensating for the larger imbalance.
Increase in Functional Ca
2+
Channels in Cerebral Smooth Muscle With Renal Hypertension
Authors: Simard J. Marc , Li Xing , Tewari Kirti
Published: June 2012

Abstract
—The hypothesis that availability of functional Ca
2+
channels in vascular smooth muscle is augmented in hypertension was tested in basilar artery cells from Wistar rats exhibiting stable systolic blood pressure (BP
sys
) for 2 to 11 weeks after partial renal artery ligation (Goldblatt 2-kidney 1-clip [2K1C] model). Cells were freshly isolated and patch-clamped using a nystatin–perforated patch method. BP
sys
ranged from 110 to 280 mm Hg and correlated with normalized kidney mass. Macroscopic current-voltage curves were fit to a Boltzmann function to obtain maximum conductance (g
max
), steepness and midpoint potential for the voltage dependence of activation (k and E
1/2
, respectively), and extrapolated reversal potential for the chord conductance (E
rev
). Linear regression of normalized conductance (ng
max
=g
max
/cell capacitance) versus BP
sys
for 103 cells indicated a strong relationship, with a slope of 0.0019 nS · pF
−1
· mm Hg
−1
(
P
<0.0001). Similar analysis of data from 35 other cells exposed to 500 nmol/L Bay K 8644 gave a slope of 0.0041 nS · pF
−1
· mm Hg
−1
(
P
=0.001). Voltage-dependent parameters, k, E
1/2
, and E
rev
, were not significantly related to BP
sys
. Single-channel measurements in cell-attached patches revealed that the number of channels in 32 patches was significantly related to BP
sys
(
P
=0.0024) but that slope conductance, open dwell times at 0 mV, and distribution between 2 open states were not. Finally, in a subgroup of 61 cells from animals made hypertensive (180 mm Hg<BP
sys
<200 mm Hg) for ≈1/2 to 6 weeks, we found that elevation of ng
max
depended on duration of hypertension (
P
=0.003), with no elevation at ≈1/2 week. We conclude that in the 2K1C model, availability of functional Ca
2+
channels increases with BP
sys
with no change in channel properties and that measurable BP
sys
elevation occurs before the increase in functional channels.
Transfer of Wild-Type p53 Gene Effectively Inhibits Vascular Smooth Muscle Cell Proliferation In Vitro and In Vivo
Authors: Yonemitsu Yoshikazu , Kaneda Yasufumi , Tanaka Shinji , Nakashima Yutaka , Komori Kimihiro , Sugimachi Keizo , Sueishi Katsuo
Published: June 2012

Abstract
—Wild-type p53 (wt-p53), a key protein in cell cycle regulation, inactivates the G
1
cyclins through direct activation of p21
Waf-1/Cip-1/Sdi-1
. Persistent vascular smooth muscle cell (VSMC) proliferation following vascular interventions hinders the benefits of these therapeutics. Using the hemagglutinating virus of Japan/liposome–mediated gene transfer method, we examined the inhibitory effect of overexpression of exogenous wt-p53 on VSMC proliferation in vitro and in vivo. We assessed the proliferative activity of human p53 cDNA–transduced bovine VSMCs by DNA synthesis assay, flow cytometry, and cell proliferation assay. p53 gene transfer reduced thymidine incorporation of VSMCs stimulated by platelet-derived growth factor-BB (
P
<.001). The p53-transduced VSMCs underwent synthetic phase depletion (mean, 8.02% versus 33.7% of control;
P
<.001) and transient G
2
/M accumulation 2 days after gene transfection, and in almost all cells, G
1
arrest occurred (mean, 92.6% versus 79.3% of control;
P
<.001) 5 days later. The wt-p53 gene transfection also inhibited the VSMC proliferation (
P
<.001) with no detectable induction of apoptosis. Cell death of p53-transduced VSMCs was induced only by additional treatment with an apoptosis-stimulating reagent, doxorubicin. The verification of apoptosis was made by DNA ladder, flow cytometry, and electron microscopy. In vivo transfection of p53 cDNA inhibited neointimal formation after balloon injury in rabbit carotid arteries, without apoptotic stimuli (P<.01). Thus, overexpression of the p53 gene in the injured arterial wall inhibits the proliferation of VSMCs in vitro and in vivo. This novel concept, including not only exogenous but also endogenous p53 overexpression in the vessel wall, may be one approach worth exploring in the treatment of patients with restenosis occurring after vascular interventions.
Operative Condition–Dependent Response of Cardiac ATP-Sensitive K
+
Channels Toward Sulfonylureas
Authors: Brady Peter A. , Alekseev Alexey E. , Terzic Andre
Published: June 2012

Abstract
—A defining property of ATP-sensitive K
+
(K
ATP
) channels is inhibition by sulfonylurea drugs, yet the response of cardiac K
ATP
channels toward sulfonylureas during myocardial ischemia is not consistent. Altered channel sensitivity toward sulfonylureas has, in part, been ascribed to antagonism by cytosolic nucleotide diphosphates, although the mechanism of interaction remains unclear. Herein, in inside-out patches excised from cardiomyocytes, we observed a dual response of K
ATP
channels toward the sulfonylurea drug, glyburide, in the presence of cytosolic UDP. Specifically, glyburide failed to inhibit spontaneous K
ATP
channel activity in the presence of UDP but inhibited UDP-induced channel activity after rundown of spontaneous channel openings. Such behavior of K
ATP
channels cannot be explained by differences in the level of channel activity or by UDP-induced displacement of glyburide. Rather, the dual response toward the sulfonylurea could be attributed to a property of K
ATP
channels to switch between operative conditions (spontaneous versus UDP-induced) each associated with a distinct responsiveness toward ligands. Conversion of post-rundown K
ATP
channels to the spontaneously operative channel condition, by Mg-ATP, restored the ability of UDP to antagonize the inhibitory action of glyburide lost after rundown, suggesting that the response of the channel to glyburide is phosphorylation dependent. The existence of distinct operative conditions of cardiac K
ATP
channels could be the basis for the inconsistent response of the channel toward sulfonylurea drugs and should be considered when sulfonylureas are used to implicate the opening of K
ATP
channels in the myocardium.
Gene Therapy for Restenosis
Authors: Baek Sanghong , March Keith L.
Published: June 2012

Abstract
—Intensive work over the past decade has been directed to the study of vascular gene transfer as an approach to the unresolved problem of restenosis. This effort has resulted in a significant foundation of knowledge relative to the activities of potentially therapeutic gene products as well as the capabilities and limitations of vector systems and mechanical delivery modalities available for effecting the vascular expression of these gene products. In several instances, significant progress has been made by experiments highlighting unexpected difficulties and the need for more comprehensive understanding. It is thus now possible to clearly define and address specific challenges that must be overcome in order to make feasible progress from the preclinical to the clinical arena. The key challenges at present appear to include the evolution of clinically practical delivery methods that meet the kinetic requirements of achieving efficient gene transduction and the availability of vectors that maximize efficiency while minimizing undesirable host responses. Emerging data suggest that approaches to solving each of these issues may have recently been developed. Basic research evaluating these new delivery mechanisms and molecular vectors is essential to establish their true potential for use in the clinical arena.
Gene Therapy for Restenosis
Authors: DeYoung Mary Beth , Dichek David A.
Published: June 2012

Abstract
—The application of gene therapy techniques to the clinical problem of coronary restenosis has generated tremendous attention and enthusiasm. Use of gene transfer technology to prevent a common intractable illness would represent a watershed event for human gene therapy. However, the time is not yet right to initiate gene therapy trials for restenosis. The biology of restenosis is incompletely understood, catheter-based gene delivery is poorly adapted to the coronary circulation, and current gene transfer vectors are ill-suited for safe and effective gene delivery to the coronary artery wall. Basic research designed to overcome these obstacles is currently more appropriate than the initiation of clinical trials.
Role of Cytoplasmic Tail of the Type 1A Angiotensin II Receptor in Agonist– and Phorbol Ester–Induced Desensitization
Authors: Tang Hua , Guo Deng Fu , Porter James P. , Wanaka Yoshio , Inagami Tadashi
Published: June 2012

Abstract
—To investigate mechanisms underlying the agonist-induced desensitization of the type 1A angiotensin II receptor (AT
1A
-R), we have stably expressed in Chinese hamster ovary (CHO) cells the wild-type receptor and truncated mutants lacking varying lengths of the cytoplasmic tail. Assay of inositol 1,4,5-trisphosphate (IP
3
) formation in response to agonist demonstrated that the truncated mutants T318, T328, and T348 lacking the last 42, 32, or 12 amino acid residues, respectively, couple with G
q
protein with an efficiency similar to that of full-length receptors, whereas coupling of G
q
protein was abolished in the T310 truncated mutant devoid of the carboxyl-terminal 50 amino acids. Exposure of CHO/AT
1A
-R cells expressing the wild-type AT
1A
-R to angiotensin II resulted in rapid and dose-dependent homologous desensitization of receptor-mediated IP
3
formation, which was independent of the receptor internalization. Mastoparan, an activator of G protein–coupled receptor kinase (GRK), induced desensitization of the AT
1A
-R. The agonist-induced desensitization of the receptor was largely prevented by heparin, a potent inhibitor of GRK, whereas it was only partially attenuated by a protein kinase C (PKC)-specific inhibitor. The homologous or heterologous desensitization of the receptor was greatly impaired in the truncated mutants T318 and T328, lacking the Ser/Thr-rich (13 or 12 Ser/Thr residues) cytoplasmic tail of the AT
1A
-R. Deletion of the last two Ser residues, including one PKC consensus site in the receptor tail, prevented only phorbol 12-myristate 13-acetate–induced desensitization by 30%. Moreover, we found an agonist-induced translocation of a heparin-sensitive kinase activity. The angiotensin II–stimulated heparin-sensitive kinase could phosphorylate a thioredoxin fusion protein containing the entire AT
1A
-R cytoplasmic tail (N
295
to E
359
), which lacks consensus phosphorylation sites for GRK1, GRK2, and GRK3. The heparin-sensitive kinase may not be GRK2, GRK3, or GRK6 expressed in CHO/AT
1A
-R cells, since angiotensin II did not induce translocation of these receptor kinases. Potential Ser/Thr phosphorylation sites located between S
328
and S
347
in the cytoplasmic tail of AT
1A
-R seem to play a critical role in the heterologous and homologous desensitization of the receptor. A heparin-sensitive kinase other than GRK2, GRK3, or GRK6 may be involved in the agonist-induced homologous desensitization of the AT
1A
-R.
Oscillatory Shear Stress Stimulates Adhesion Molecule Expression in Cultured Human Endothelium
Authors: Chappell David C. , Varner Signe E. , Nerem Robert M. , Medford Russell M. , Alexander R. Wayne
Published: June 2012

Abstract
—Low and oscillatory shear stresses are major features of the hemodynamic environment of sites opposite arterial flow dividers that are predisposed to atherosclerosis. Atherosclerosis is a focal inflammatory disease characterized initially by the recruitment of mononuclear cells into the arterial wall. The specific characteristics of the hemodynamic environment that facilitate the generation of arterial inflammatory responses in the presence of, for example, hyperlipidemia are unknown. We show here that prolonged oscillatory shear stress induces expression of endothelial cell leukocyte adhesion molecules, which are centrally important in mediating leukocyte localization into the arterial wall. Vascular cell adhesion molecule-1 was upregulated an average 9-fold relative to endothelial monolayers in static culture. Intercellular adhesion molecule-1 and E-selectin exhibited 11-fold and 7.5-fold increases, respectively. Upregulation of these adhesion molecules was associated with enhanced monocyte adherence. Cytokine stimulation of surface vascular cell adhesion molecule-1 was maximally induced after 6 and 8 hours of cytokine incubation. Oscillatory shear stress for these time periods elicited respective vascular cell adhesion molecule-1 levels of 16% and 30% relative to those observed for cytokine stimulation. Surface intercellular adhesion molecule-1 induction by cytokine stimulation for 24 hours was found to be approximately five times the level detected after 24 hours of oscillatory shear stress. Experiments performed in the presence of the antioxidant
N
-acetylcysteine demonstrated that the expression of vascular cell adhesion molecule-1 could be almost totally abolished, whereas that of intercellular adhesion molecule-1 was typically reduced by ≈70%. These results imply that oscillatory shear stress per se is sufficient to stimulate mononuclear leukocyte adhesion and, presumptively, migration into the arterial wall. These results further indicate that atherosclerotic lesion initiation is likely related, at least in part, to unique signals generated by oscillatory shear stress and that the mechanism of upregulation is, to some extent, redox sensitive.
Expression and Regulation of Adhesion Molecules in Cardiac Cells by Cytokines
Authors: Kacimi Rachid , Karliner Joel S. , Koudssi Farid , Long Carlin S.
Published: June 2012

Abstract
—Adhesion molecules mediate inflammatory myocardial injury after ischemia/reperfusion. Cytokine release and hypoxia are features of acute ischemia that may influence expression of these molecules. Accordingly, we studied intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM) responses to cytokines and acute hypoxia in cultured myocardial cells. Northern blot analysis and immunoassay showed that the proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α stimulated concentration-dependent increases in ICAM and VCAM mRNA and protein. In both cardiac myocytes and fibroblasts, pretreatment with a specific inhibitor of nuclear transcription factor-κB (NF-κB) prevented cytokine induction of both molecules. We also found that inhibition of tyrosine kinase and p38/RK (stress-activated protein kinase) pathways prevented IL-1β–induced ICAM and VCAM protein synthesis, whereas extracellular signal–regulated protein kinase (ERK1/ERK2) inhibition did not. Neither hypoxia (0% O
2
for 6 hours) alone nor hypoxia/reoxygenation had any significant effect on ICAM and VCAM mRNA. However, hypoxia did enhance IL-1β–induced ICAM mRNA expression in myocytes. As a possible mechanism of this synergistic action on CAM expression, hypoxia induced a time-dependent increase in the DNA binding activity of both NF-κB and activator protein-1 (AP-1), two transcription factors important for cell adhesion molecule expression. In contrast to the enhanced ICAM mRNA induced by IL-1β during hypoxia, however, protein levels for this adhesion molecule were unchanged beyond IL-1β–stimulated levels, suggesting posttranscriptional and/or posttranslational control mechanisms. We conclude that cytokines regulate ICAM and VCAM mRNA and protein in both cardiac myocytes and fibroblasts. Furthermore, adhesion molecule induction requires translocation of at least two transcription factors, NF-κB and AP-1.
Angiotensin II Potentiates Vascular Endothelial Growth Factor–Induced Angiogenic Activity in Retinal Microcapillary Endothelial Cells
Authors: Otani Atsushi , Takagi Hitoshi , Suzuma Kiyoshi , Honda Yoshihito
Published: June 2012

Abstract
—Angiotensin II (Ang II) plays a role in the development of many vascular diseases. In the present study, we have investigated the effect of Ang II on vascular endothelial growth factor (VEGF) receptor expression and VEGF-induced angiogenic activity in bovine retinal microcapillary endothelial cells (BRECs). Ang II induced a significant increase of kinase domain–containing receptor/total liver kinase (KDR/Flk-1) mRNA in a time- and dose-dependent manner, with a maximal 4.3±0.8-fold increase after a 4-hour stimulation. Ang II increased the rate of KDR gene transcription by 5.4-fold, whereas the half-life of KDR mRNA was not increased significantly. The increase depended partially on new protein synthesis. The Ang II–induced KDR mRNA increase was inhibited by either [Sar
1
,Ile
8
]angiotensin or angiotensin type 1 receptor antagonists but was not significantly altered by angiotensin type 2 receptor antagonists. The PKC inhibitor reduced Ang II–induced KDR mRNA expression by 70±15%. The tyrosine kinase inhibitor reduced the Ang II– and phorbol 12-myristate 13-acetate–induced KDR mRNA increases by 35±8% and 44±26%, respectively. Ang II increased by 3.1-fold the
35
S-labeled KDR/Flk-1 immunoprecipitated by a specific antibody to KDR/Flk-1. Scatchard analysis demonstrated that Ang II induced a significant increase of binding sites without changing binding affinity. Ang II enhanced VEGF-induced cell growth and tube formation. Ang II itself had no effect on cell growth, tube formation, or mRNA levels of VEGF and
tms
-like tyrosine kinase (Flt-1) in BRECs. These findings suggest that Ang II might potentiate VEGF-induced angiogenic activity through an increase of the VEGF receptor KDR/Flk-1.
Cooperative Interactions Between RB and p53 Regulate Cell Proliferation, Cell Senescence, and Apoptosis in Human Vascular Smooth Muscle Cells From Atherosclerotic Plaques
Authors: Bennett Martin R. , Macdonald Kirsty , Chan Shiu-Wan , Boyle Joseph J. , Weissberg Peter L.
Published: June 2012

Abstract
—Compared with vascular smooth muscle cells (VSMCs) from normal vessels, VSMCs from human atherosclerotic plaques proliferate more slowly, undergo earlier senescence, and demonstrate higher levels of apoptosis in culture. The tumor suppressor genes p105
RB
(retinoblastoma, acting through the E2F transcription factor family) and p53 regulate cell proliferation, cell senescence, and apoptosis in many cell types. We have therefore determined whether these stable growth properties of plaque VSMCs reflect altered activity of RB and/or p53. VSMCs were derived from coronary atherectomies or from normal coronary arteries from transplant recipients. Compared with normal VSMCs, plaque VSMCs showed a higher ratio of the active (hypophosphorylated) to the inactive (phosphorylated) form of RB and a lower level of E2F transcriptional activity. Cells were stably transfected with retrovirus constructs that inhibited RB or p53 alone or in combination. Suppression of RB alone increased rates of cell proliferation and apoptosis and inhibited cell senescence in normal VSMCs. Suppression of p53 and RB together had similar effects but, additionally, resulted in immortalization of normal VSMC cultures. In contrast, inhibition of RB binding to E2F or ectopic expression of E2F-1 in plaque VSMCs induced massive apoptosis, which required suppression of p53 to rescue cells. Suppression of RB and p53 together increased cell proliferation and delayed senescence but failed to immortalize plaque VSMCs. Inhibition of p53 alone had minimal effects on plaque VSMCs but increased the lifespan of normal VSMCs. We conclude that human plaque VSMCs have slower rates of cell proliferation and earlier senescence than do cells from normal vessels because of a defect in phosphorylation of RB. Furthermore, both disruption of RB/E2F and inhibition of p53 are required for plaque VSMCs to proliferate without apoptosis. This observation may explain the relatively low level of cell proliferation and high level of apoptosis seen in VSMCs in human atherosclerotic plaques.
Regulation of Vascular Connexin43 Gene Expression by Mechanical Loads
Authors: Cowan Douglas B. , Lye Stephen J. , Langille B. Lowell
Published: June 2012

Abstract
—Vascular tissues respond to changes in the mechanical forces imposed on them with changes in vasomotor tone in the short term and with structural remodeling in the long term. Since these responses involve intercellular communication, we have investigated regulation of the gap junction proteins, connexin26 (Cx26), connexin37 (Cx37), connexin40 (Cx40), and connexin43 (Cx43), by mechanical loads. Results were compared with parallel experiments on c-
fos
and GAPDH. Twenty percent stretch of cultured vascular smooth muscle cells caused a 3-fold increase in Cx43 mRNA levels by 2 hours. Cx26 was expressed at low levels but failed to respond to stretch, and Cx37 and Cx40 were not detected. c-
fos
mRNA levels increased after 30 minutes of stretch, whereas GAPDH mRNA did not change. Protein levels of Cx43 increased by 4 hours and remained elevated for 16 hours. Nuclear run-on experiments confirmed that Cx43 and c-
fos
were transcriptionally regulated by stretch. New protein synthesis was not a requirement for the stretch-induced rise in Cx43 expression, since mRNA levels were unaffected by treatment with cycloheximide. To examine transcriptional control of Cx43, stretched and unstretched vascular smooth muscle cells were transfected with a variety of promoter-reporter gene constructs. Cx43 sequences extending from within exon 1 (+162) to −1686 in the 5′-flanking region were coupled to the chloramphenicol acetyl transferase reporter gene. Deletions from the 5′ end of these sequences differentially regulated reporter gene expression and indicated multiple potential regulatory sites. In particular, a putative activator protein-1 site at the −42 to −48 region was required for basal reporter activity. None of the promoter constructs revealed stretch sensitivity, indicating that the site of transcriptional control by stretch lies outside the −1686 to +162 region. Finally, Cx43 mRNA levels were assessed in cultured endothelial cells subjected to laminar shear stress of 15 dynes/cm
2
. Cx43 mRNA levels increased by ≈4-fold at 1 hour and remained elevated for the duration of shear force. In conclusion, both mechanical strain and fluid shear stress caused increased expression of the gap junction protein Cx43.
Fluid Shear Stress Increases the Production of Granulocyte-Macrophage Colony-Stimulating Factor by Endothelial Cells via mRNA Stabilization
Authors: Kosaki Keisuke , Ando Joji , Korenaga Risa , Kurokawa Takahide , Kamiya Akira
Published: June 2012

Abstract
—To investigate whether the production of colony-stimulating factors (CSFs) by vascular endothelial cells is regulated by hemodynamic force, we exposed cultured human umbilical vein endothelial cells (HUVECs) to controlled levels of shear stress in a flow-loading apparatus and examined changes in the production of CSFs at both the protein and mRNA level. Exposure of HUVECs to a shear stress of 15 and 25 dyne/cm
2
markedly increased the release of granulocyte-macrophage CSF (GM-CSF) detected by ELISA to 5.0 and 9.5 times, respectively, the amount released by the static controls at 24 hours, but it had no significant influence on the release of granulocyte CSF or macrophage CSF. The results of reverse transcriptase–polymerase chain reaction demonstrated that GM-CSF mRNA began to increase as early as 2 hours after initiation of 15 dyne/cm
2
shear stress and continued to increase with time, reaching a peak of about four times the control levels at 24 hours. This increase in GM-CSF mRNA levels in response to shear stress depended on protein synthesis, because it was blocked by cycloheximide. Neither nuclear run-on assay or luciferase assay using a reporter gene containing GM-CSF gene promoter showed any significant change in transcription of the GM-CSF gene even after 24-hour exposure to a shear stress of 15 dyne/cm
2
. Actinomycin D chase experiments using a competitive polymerase chain reaction showed that shear stress extended the half-life of GM-CSF mRNA from ≈23 to 42 minutes in HUVECs. These findings suggest that fluid shear stress increases the production of GM-CSF in HUVECs via mRNA stabilization.
Superoxide Anion From the Adventitia of the Rat Thoracic Aorta Inactivates Nitric Oxide
Authors: Di Wang Hui , Pagano Patrick J. , Du Yue , Cayatte Antonio J. , Quinn Mark T. , Brecher Peter , Cohen Richard A.
Published: June 2012

Abstract
—The purpose of this study was to determine whether superoxide anion is produced endogenously in the rat aortic adventitia and whether sufficient superoxide anion is produced to interfere with the response of the rat aorta to nitric oxide. Relaxation was measured in rings of the rat thoracic aorta, which were oriented so that the adventitial or luminal surface could be preferentially exposed to nitric oxide or sodium nitroprusside. To accomplish this, the rings were mounted (1) with the adventitia facing outward, (2) with the adventitia facing inward after inverting, or (3) with the adventitia facing outward after inverting twice (to control for the inverting procedure). The relaxation to nitric oxide, but not to sodium nitroprusside, was less in rings with the adventitia facing outward compared with those in which it faced inward. In contrast, the response to nitric oxide via either surface was similar when extracellular superoxide anion was scavenged with superoxide dismutase. Incubation of rings with nitro blue tetrazolium (NBT) resulted in blue formazan staining of the adventitia, and lucigenin chemiluminescence was significantly greater when detected from the adventitial compared with the intimal aspect of the artery. The reduction of NBT in intact aortic rings was 30±2 pmol · min
−1
· mg
−1
and was significantly decreased by superoxide dismutase to 19±2 pmol · min
−1
· mg
−1
and by a synthetic superoxide dismutase mimic, Euk-8, to 11±2 pmol · min
−1
· mg
−1
. The NADPH oxidase inhibitor, diphenyleneiodonium, decreased NBT reduction to 9±1 pmol · min
−1
· mg
−1
, whereas inhibitors of xanthine oxidase, mitochondrial oxidases, and nitric oxide synthase were ineffective. Immunohistochemical staining indicated the localization of NADPH oxidase proteins gp91
phox
, p22
phox
, p47
phox
, and p67
phox
almost exclusively in the adventitia of the rat aorta with no substantial staining in the media. These results indicate that NADPH oxidase located in the adventitia of rat thoracic aorta generates sufficient extracellular superoxide anion to constitute a barrier capable of inactivating nitric oxide. This study suggests that adventitial superoxide anion can play a role in the pathophysiology of the arterial wall.
Synergistic Inhibition of Vascular Smooth Muscle Cell Migration by Phosphodiesterase 3 and Phosphodiesterase 4 Inhibitors
Authors: Palmer Daniel , Tsoi Keith , Maurice Donald H.
Published: June 2012

Abstract
—Cyclic nucleotide phosphodiesterases (PDEs) hydrolyze cAMP or cGMP and terminate their signaling. Two important families of PDEs that regulate cAMP signaling in cardiovascular tissues are the cGMP-inhibited PDEs (PDE3) and the cAMP-specific PDEs (PDE4). In this study, we have used a combination of an in vitro motility assay and a sensitive method for the measurement of cAMP in order to determine the relative roles of PDE3 and of PDE4 in the regulation of cAMP-mediated inhibition of VSMC migration. Our data demonstrate that forskolin, an activator of adenylyl cyclases, causes concentration-dependent inhibition of platelet-derived growth factor–induced VSMC migration. Incubation of cultured VSMCs with a PDE4-selective inhibitor, Ro 20-1724, markedly potentiated both the antimigratory effect and the increase in cAMP caused by forskolin. Cilostamide, a PDE3-selective compound, did not affect either the antimigratory activity of forskolin or its ability to increase cAMP. Cilostamide and Ro 20-1724 interacted synergistically to potentiate the inhibition of VSMC migration by forskolin and caused a supra-additive increase in cAMP. These data are consistent with an important role for both PDE3 and PDE4 in the regulation of cAMP-mediated inhibition of VSMC migration.
Adhesion of Monocytes to Vascular Cell Adhesion Molecule-1–Transduced Human Endothelial Cells
Authors: Gerszten Robert E. , Lim Yaw-Chyn , Ding Han T. , Snapp Karen , Kansas Geoffrey , Dichek David A. , Cabañas Carlos , Sánchez-Madrid Francisco , Gimbrone Michael A. , Rosenzweig Anthony , Luscinskas Francis W.
Published: June 2012

Abstract
—To study the role of vascular cell adhesion molecule-1 (VCAM-1) in monocyte recruitment and atherogenesis, we constructed a recombinant adenovirus, AdRSVrVCAM-1, carrying the rabbit VCAM-1 cDNA. We have previously shown that AdRSVrVCAM-1–transduced human umbilical vein endothelial cells (HUVECs) support the adhesion of CD4
+
CD45RO
+
memory T lymphocytes under laminar flow conditions. We now demonstrate that AdRSVrVCAM-1–transduced HUVECs support the adhesion of peripheral blood monocytes at a shear stress of ≤1.5 dyne/cm
2
. Although VCAM-1 supported only firm adhesion of lymphocytes, it was able to mediate monocyte rolling, firm adhesion, and transmigration when expressed in the context of otherwise unactivated vascular endothelium. VCAM-1–transduced HUVECs supported the adhesion of as many as 4-fold more monocytes than T cells under laminar flow. The greater monocyte adhesion was explained at least in part by leukocyte-leukocyte interactions (secondary adhesions), which were not seen with T cells. These secondary monocyte interactions were specifically blocked by monoclonal antibodies to L-selectin and P-selectin glycoprotein ligand-1. These data demonstrate that VCAM-1 expressed in the context of unactivated vascular endothelium supports the adhesion of the leukocyte populations present in atherosclerotic plaque and may contribute to the predominance of monocytes over lymphocytes.
Role of Nitric Oxide in the Angiogenic Response In Vitro to Basic Fibroblast Growth Factor
Authors: Babaei Saeid , Teichert-Kuliszewska Krystyna , Monge Juan-Carlos , Mohamed Farida , Bendeck Michelle P. , Stewart Duncan J.
Published: June 2012

Abstract
—Angiogenesis is a complex process that involves the activation of quiescent endothelial cells (ECs) to a proliferative and migratory phenotype and, subsequently, their redifferentiation to form vascular tubes. We hypothesized that NO contributes to angiogenesis by terminating the proliferative action of angiogenic growth factors and initiating a genetic program of EC differentiation. Human umbilical vein ECs (HUVECs) and calf pulmonary artery ECs (CPAECs) were grown directly on plastic dishes or on three-dimensional fibrin matrices. In the absence of fibrin, treatment with NO-donor compounds, such as
S
-nitroso-
N
-acetylpenicillamine (SNAP, 0.1 and 0.4 mmol/L), produced a dose-dependent inhibition of proliferation in both cell lines, whereas the inhibition of endogenous NO production using
N
G
-nitro-
l
-arginine methyl ester (L-NAME, 1 mmol/L) or
N
G
-monomethyl-
l
-arginine (L-NMMA, 1 mmol/L) significantly increased proliferation of the CPAECs. The addition of basic fibroblast growth factor (bFGF, 30 ng/mL) increased the expression of endothelial NO synthase mRNA and the production of NO in both cell types when cultured on three-dimensional fibrin gels and produced profound morphological changes characterized by the appearance of extensive capillary-like vascular structures and the loss of EC monolayers. These changes were quantified by measuring total tube length per low-power field (×100), and a differentiation index was derived using the ratio of tube length over area covered by residual EC monolayer. In the absence of additional angiogenic factors, the differentiation index was low for both HUVECs and CPAECs (control, 1.16±0.19 and 2.07±0.87, respectively). Treatment with bFGF increased the differentiation index significantly in both cell types (10.59±2.03 and 20.02±5.01 for HUVECs and CPAECs, respectively;
P
<.05 versus control), and the addition of SNAP (0.4 mmol/L) mimicked the angiogenic response to bFGF (8.57±1.34 and 12.20±3.49 for HUVECs and CPAECs, respectively;
P
<.05 versus control). Moreover, L-NAME inhibited EC tube formation in response to bFGF in a dose-response manner, consistent with a role of endogenous NO production in EC differentiation in this angiogenic model. These findings suggest that NO may act as a crucial signal in the angiogenic response to bFGF, terminating the proliferative actions of angiogenic growth factors and promoting EC differentiation into vascular tubes.
A 17mer Peptide Interferes With Acidification-Induced Uncoupling of Connexin43
Authors: Calero Guillermo , Kanemitsu Martha , Taffet Steven M. , Lau Alan F. , Delmar Mario
Published: June 2012

Abstract
—Structure/function analysis shows that the carboxyl terminal (CT) domain of connexin43 (Cx43) is essential for the chemical regulation of cell-cell communication. Of particular interest is the region between amino acids 260 and 300. Structural preservation of this region is essential for acidification-induced uncoupling (ie, pH gating). In this study, we report data showing that a 17mer peptide of the same sequence as amino acids 271 to 287 of Cx43 (CSSPTAPLSPMSPPGYK) can prevent pH gating of Cx43-expressing oocytes. Experiments were carried out in pairs of
Xenopus
oocytes previously injected with connexin38 antisense and expressing wild-type Cx43. Junctional conductance was measured electrophysiologically. pH
i
was determined from the light emission of the proton-sensitive dye dextran-seminaphtho- rhodafluor. Intracellular acidification was induced by superfusion with a bicarbonate-buffered solution gassed with a progressively increasing concentration of CO
2
. Injection of water alone into both oocytes of a Cx43-expressing pair or injection of a peptide from region 321 to 337 of Cx43 did not modify pH sensitivity. However, injection of a polypeptide corresponding to amino acids 241 to 382 of Cx43 interfered with the ability of gap junctions to close on acidification. Similar results were obtained when a 17mer peptide (region 271 to 287) was injected into both oocytes of the pair. Normal Cx43 pH gating was observed if (1) the amino acid sequence of the 17mer peptide was scrambled or (2) the N and the C ends of the 17mer peptide were not included in the sequence. This is the first demonstration of a molecule that can interfere with the chemical regulation of connexin channels in a cell pair. The data may lead to the development of small molecules that can be used in Cx43-expressing multicellular preparations to study the role of gap junction regulation in normal as well as diseased states.
Cloning and Characterization of α1H From Human Heart, a Member of the T-Type Ca
2+
Channel Gene Family
Authors: Cribbs Leanne L. , Lee Jung-Ha , Yang Jie , Satin Jonathan , Zhang Yi , Daud Asif , Barclay Jane , Williamson Magali P. , Fox Margaret , Rees Michele , Perez-Reyes Edward
Published: June 2012

Abstract
—Voltage-activated Ca
2+
channels exist as multigene families that share common structural features. Different Ca
2+
channels are distinguished by their electrophysiology and pharmacology and can be classified as either low or high voltage–activated channels. Six α1 subunit genes cloned previously code for high voltage–activated Ca
2+
channels; therefore, we have used a database search strategy to identify new Ca
2+
channel genes, possibly including low voltage–activated (T-type) channels. A novel expressed sequence–tagged cDNA clone of α1G was used to screen a cDNA library, and in the present study, we report the cloning of α1H (or Ca
v
T.2), a low voltage–activated Ca
2+
channel from human heart. Northern blots of human mRNA detected more α1H expression in peripheral tissues, such as kidney and heart, than in brain. We mapped the gene,
CACNA1H
, to human chromosome 16p13.3 and mouse chromosome 17. Expression of α1H in HEK-293 cells resulted in Ca
2+
channel currents displaying voltage dependence, kinetics, and unitary conductance characteristic of native T-type Ca
2+
channels. The α1H channel is sensitive to mibefradil, a nondihydropyridine Ca
2+
channel blocker, with an IC
50
of 1.4 μmol/L, consistent with the reported potency of mibefradil for T-type Ca
2+
channels. Together with α1G, a rat brain T-type Ca
2+
channel also cloned in our laboratory, these genes define a unique family of Ca
2+
channels.
Involvement of IsK-Associated K
+
Channel in Heart Rate Control of Repolarization in a Murine Engineered Model of Jervell and Lange-Nielsen Syndrome
Authors: Drici Milou-Daniel , Arrighi Isabelle , Chouabe Christophe , Mann Jeffrey R. , Lazdunski Michel , Romey Georges , Barhanin Jacques
Published: June 2012

Abstract
—The Jervell and Lange-Nielsen (JLN) syndrome affects the human cardioauditory system, associating a profound bilateral deafness with an abnormally long QT interval on the ECG. It results from mutations in
KVLQT1
and
ISK
genes that encode the 2 subunits forming the K
+
channel responsible for the cardiac and inner ear slowly activating component of the delayed rectifier K
+
current (
I
Ks
). A JLN mouse model that presents typical inner ear defects has been created by knocking out the
isk
gene (
isk
−/−). This study specifically reports on the cardiac phenotype counterpart, determined in the whole animal and at mRNAs and cellular levels. Surface ECG recordings of
isk
−/− mice showed a longer QT interval at slow heart rates, a paradoxical shorter QT interval at fast heart rates, and an overall exacerbated QT–heart rate adaptation compared with wild-type (WT) mice. A 300-ms increase in the heart rate cycle length induces a 309±21% increase in the QT duration of the WT mice versus a 500±50% in
isk
−/− mice (
P
<0.001). It is concluded that the
isk
gene product and/or
I
Ks
, when present, blunts the QT adaptation to heart rate variations and that steeper QT-RR relationships reflect a greater susceptibility to arrhythmias in patients lacking
I
Ks
.
Pathophysiological Role of Angiotensin II Type 2 Receptor in Cardiovascular and Renal Diseases
Authors: Matsubara Hiroaki
Published: June 2012

Abstract
—Since the discovery of nonpeptidic ligands, the receptors for angiotensin (Ang) II have been classified into 2 subtypes (Ang II type 1 receptor [AT
1
-R] and Ang II type 2 receptor [AT
2
-R]). AT
1
-R mediates most of the cardiovascular actions of Ang II. AT
2
-R is expressed at very high levels in the developing fetus. Its expression is very low in the cardiovascular system of the adult. The expression of AT
2
-R can be modulated by pathological states associated with tissue remodeling or inflammation. In failing hearts or neointima formation after vascular injury, AT
2
-R is reexpressed in cells proliferating in interstitial regions or neointima and exerts an inhibitory effect on Ang II-induced mitogen signals or synthesis of extracellular matrix proteins, resulting in attenuation of the tissue remodeling. An extreme form of cell growth inhibition ends in programmed cell death, and this process, which is initiated by the withdrawal of growth factors, is also enhanced by AT
2
-R. Cardiac myocyte- or vascular smooth muscle–specific mice that overexpress AT
2
-R display an inhibition of Ang II-induced chronotropic or pressor actions, suggesting the role of AT
2
-R on the activity of cardiac pacemaker cells and the maintenance of vascular resistance. AT
2
-R also activates the kinin/nitric oxide/cGMP system in the cardiovascular and renal systems, resulting in AT
2
-R–mediated cardioprotection, vasodilation, and pressure natriuresis. These effects, transmitted by AT
2
-R, are mainly exerted by stimulation of protein tyrosine or serine/threonine phosphatases in a Gi protein–dependent manner. The expression level of AT
2
-R is much higher in human hearts than in rodent hearts, and the AT
2
-R–mediated actions are likely enhanced, especially by clinical application of AT
1
-R antagonists. Thus, in this review, the regulation of AT
2
-R expression, its cellular localization, its pathological role in cardiovascular and kidney diseases, and pharmacotherapeutic effects of AT
2
-R stimulation are discussed.
Individual Gap Junction Plaques Contain Multiple Connexins in Arterial Endothelium
Authors: Yeh Hung-I , Rothery Stephen , Dupont Emmanuel , Coppen Steven R. , Severs Nicholas J.
Published: June 2012

Abstract
—Gap-junctional intercellular communication in endothelial cells is implicated in the coordination of growth, migration, and vasomotor responses. Up to 3 connexin types, connexin40 (Cx40), Cx37, and Cx43 may be expressed in vascular endothelium according to vascular site, species, and physiological conditions. To establish how these connexins are organized at the level of the individual endothelial gap junction, we used affinity-purified connexin-specific antibodies raised in 3 different species to permit double and triple immunolabeling in combination with confocal and electron microscopy. Using HeLa cells transfected with Cx37 and Cx40 for characterization, the anti-Cx37 antibody (raised in rabbit) and the anti-Cx40 antibody (raised in guinea pig) were shown to recognize single bands of 37 and 40 kDa, respectively, on Western blots and to give prominent punctate labeling at the cell borders, specifically in the corresponding transfectant. By applying these antibodies together with mouse monoclonal anti-Cx43 for double and triple immunofluorescence labeling at confocal microscopy, rat aortic and pulmonary arterial endothelia were found to express all 3 connexin types, whereas coronary artery endothelium expressed Cx40 and Cx37 but lacked Cx43. High-resolution en face confocal viewing of the aortic endothelium after double labeling demonstrated frequent colocalization of connexins, with distinct variation in the expression pattern within a given cell, where it made contact with different neighbors. Triple immunogold labeling at the electron-microscopic level revealed that aortic endothelial gap junctions commonly contain all 3 connexin types. This represents the first definitive demonstration of any cell type in vivo expressing 3 different connexins organized within the same gap-junctional plaque.
Effects of the Diuretic Agent Indapamide on Na
+
, Transient Outward, and Delayed Rectifier Currents in Canine Atrial Myocytes
Authors: Lu Yanjie , Yue Lixia , Wang Zhiguo , Nattel Stanley
Published: June 2012

Abstract
—The diuretic agent indapamide has been reported to block the slow component of the delayed rectifier K
+
current (
I
Ks
) without altering the rapid component (
I
Kr
) or the inward rectifier current and has been used as a pharmacological probe for
I
Ks
; however, the effects of indapamide on Na
+
(
I
Na
), L-type Ca
2+
(
I
Ca
), and transient outward K
+
(
I
to
) currents have not been determined. We applied tight-seal, whole-cell, patch-clamp techniques to assess the effects of indapamide on
I
Na
,
I
to
,
I
Ca
, and
I
Ks
in canine atrial myocytes. Indapamide inhibited
I
Na
,
I
to
, and
I
Ks
in a concentration-dependent and reversible way, without altering
I
Ca
. Block increased with depolarization, with the 50% blocking concentration (EC
50
) decreasing from 129±26 μmol/L (at −60 mV) to 79±17 μmol/L (at −10 mV) for
I
Na
, from 174±19 μmol/L (at +10 mV) to 98±7 μmol/L (at +60 mV) for
I
to
, and from 148±28 μmol/L (at +10 mV) to 86±18 μmol/L (at +60 mV) for
I
Ks
. Significant inhibition was seen at concentrations as low as 10 μmol/L for all 3 currents. In addition, indapamide effectively inhibited the ultrarapid delayed rectifier current in a voltage-independent way, with an EC
50
of 138±7 μmol/L at +10 mV. Standard microelectrode experiments showed the effects of indapamide on the action potential to be consistent with the ionic actions seen. We conclude that in addition to its well-recognized
I
Ks
-blocking action, indapamide also inhibits
I
Na
and
I
to
effectively and with similar potency. Thus, indapamide is not a reliable pharmacological probe with which to study the specific effects of
I
Ks
blockade, and
I
Na
and
I
to
block may contribute to the potential profile of cardiac actions of the compound.
P2X
7
Receptor Activation–Induced Contraction and Lysis in Human Saphenous Vein Smooth Muscle
Authors: Cario-Toumaniantz Chrystelle , Loirand Gervaise , Ladoux Annie , Pacaud Pierre
Published: June 2012

Abstract
—In cutaneous veins where purinergic neurotransmission is more prominent compared with in deep vessels, physiological and pathological roles of nerve-released ATP have been described. Neuronally released ATP has been reported to act through activation of unidentified ionotropic P2X receptor(s). This study analyzed P2X receptor subtypes expressed in human saphenous vein smooth muscle and their physiological functions. Transcripts for both hP2X
1
receptors, already identified in other smooth muscles, and, surprisingly, hP2X
7
receptors known to be responsible for the cytotoxic effect of ATP in macrophages were detected by Northern blot analysis in total RNA from saphenous vein smooth muscle. ATP and other P2X receptor agonists [αβ-methylene-ATP, 2-methylthio-ATP, and 2′,3′-(4-benzoyl)benzoyl-ATP] dose-dependently contracted venous rings, but the contraction induced by 2-methylthio-ATP was more transient than that evoked by the other P2X agonists. The effect of hP2X
1
agonists involved the activation of a rapidly desensitizing cation current recorded in freshly isolated myocytes. The action of hP2X
7
receptor agonists was related to a maintained nondesensitizing cation current. In addition, hP2X
7
receptor activation formed membrane pores that were permeable to large molecules. hP2X
1
and hP2X
7
receptors coexpressed in COS cells did not associate to form heteromultimers. Our data indicate that both hP2X
1
and hP2X
7
receptors are expressed as 2 separated populations of channels in human saphenous vein myocytes and are involved in ATP-induced tension. We suggest that cell lysis consequent to hP2X
7
receptor–induced pore formation contributes to the disorganization and decrease in the amount of contractile myocytes in the media of varicose veins.