bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of carbon monoxide and heme oxygenase inhibitors in cerebral vessels of rats and mice
Authors: Andresen Jon J. , Shafi Nadeem I. , Durante William , Bryan Robert M.
Published: February 2006

Carbon monoxide (CO) has been postulated to be a signaling molecule in many tissues, including the vasculature. We examined vasomotor responses of adult rat and mouse cerebral arteries to both exogenously applied and endogenously produced CO. The diameter of isolated, pressurized, and perfused rat middle cerebral arteries (MCAs) was not altered by authentic CO (10−6to 10−4M). Mouse MCAs, however, dilated by 21 ± 10% at 10−4M CO. Authentic nitric oxide (NO·, 10−10to 10−7M) dilated both rat and mouse MCAs. At 10−8M NO·, rat vessels dilated by 84 ± 4%, and at 10−7M NO·, mouse vessels dilated by 59 ± 9%. Stimulation of endogenous CO production through heme oxygenase (HO) with the heme precursor δ-aminolevulinic acid (10−10to 10−4M) did not dilate the MCAs of either species. The metalloporphyrin HO inhibitor chromium mesoporphyrin IX (CrMP) caused profound constriction of the rat MCA (44 ± 2% at 3 × 10−5M). Importantly, this constriction was unaltered by exogenous CO (10−4M) or CO plus 10−5M biliverdine (both HO products). In contrast, exogenous CO (10−4M) reversed CrMP-induced constriction in rat gracilis arterioles. Control mouse MCAs constricted by only 3 ± 1% in response to 10−5M CrMP. Magnesium protoporphyrin IX (10−5M), a weak HO inhibitor used to control for nonspecific effects of metalloporphyrins, also constricted the rat MCA to a similar extent as CrMP. We conclude that, at physiological concentrations, CO is not a dilator of adult rodent cerebral arteries and that metalloporphyrin HO inhibitors have nonspecific constrictor effects in rat cerebral arteries.
In vivo recording of adult zebrafish electrocardiogram and assessment of drug-induced QT prolongation
Authors: Milan David J. , Jones Ian L. , Ellinor Patrick T. , MacRae Calum A.
Published: February 2006

In the last decade the zebrafish has become a major model organism for the study of development and organogenesis. To maximize the experimental utility of this organism, it will be important to establish methods for adult phenotyping. We previously proposed that the embryonic zebrafish may be useful in high-throughput screening for drug-induced cardiotoxicity. We now describe a method for the reproducible recording of the adult zebrafish ECG and illustrate its application in the investigation of QT-prolonging drugs. Zebrafish ECGs were obtained by inserting two needle electrodes through the ventral epidermis. Fish were perfused orally, and motion artifacts were eliminated with a paralytic dose of μ-conotoxin GIIIB. Test compounds were delivered via the perfusion system. Without a means of hydration and oxygenation, the fish succumb rapidly. The use of a perfusion system allowed stable recording for >6 h. Baseline conduction intervals were as follows: PR, 66 ms (SD 14); QRS, 34 ms (SD 11); QT, 242 ms (SD 54); and R-R, 398 ms (SD 77). The known QT-prolonging agents astemizole, haloperidol, pimozide, and terfenadine caused corrected QT increases of 18% (SD 9), 16% (SD 11), 17% (SD 9), and 11% (SD 6), respectively. The control drugs clonidine, penicillin and propranolol did not prolong the corrected QT interval. In conclusion, perfusion and muscular paralysis allows stable, low-noise recording of zebrafish ECGs. Agents known to cause QT prolongation in humans caused QT prolongation in fish in each case. The development of rigorous tools for the phenotyping of adult zebrafish will complement the high-throughput assays currently under development for embryonic and larval fish.
Effects of hsp90 binding inhibitors on sGC-mediated vascular relaxation
Authors: Yetik-Anacak Gunay , Xia Tian , Dimitropoulou Christiana , Venema Richard C. , Catravas John D.
Published: February 2006

Vascular soluble guanylate cyclase (sGC) exists in multimeric complexes with endothelial nitric oxide (NO) synthase (eNOS) and heat shock protein 90 (hsp90). Whereas disruption of hsp90-eNOS complexes clearly attenuates eNOS-dependent vascular relaxation, the contribution of sGC-hsp90 complexes to eNOS- or NO donor-dependent relaxations remains unclear. Isolated rat thoracic aortic rings were preincubated with structurally diverse hsp90 binding inhibitors, radicicol (RA) or geldanamycin (GA), or vehicle for 0.5, 1, or 15 h. Preconstricted vessels were exposed to ACh, 8-bromo-cGMP (8-BrcGMP), forskolin, or one of three NO donors: nitroglycerin (NTG), sodium nitroprusside, or spermine NONOate (SNN). Both RA and GA inhibited endothelium-dependent relaxations dose dependently. Indomethacin or the antioxidant tiron did not affect the inhibition of ACh-induced relaxations by GA. Long-term (15 h) exposure to RA inhibited all NO donor-induced relaxations; however, GA inhibited SNN-induced relaxation only. The effects of GA and RA appeared to be selective because 15-h treatment with either agent did not affect forskolin-induced relaxations and only slightly decreased 8-BrcGMP-induced relaxations. Similarly to their effects on NO-donor-induced relaxation, 15-h exposure to RA, but not to GA, decreased hsp90-bound sGC protein expression and NTG-stimulated cGMP formation in aortic rings, whereas RA more than GA reduced SNN-stimulated cGMP formation. We conclude that RA, much more so than GA, selectively inhibits sGC-dependent relaxations of aortic rings by reducing sGC expression, disrupting sGC-hsp90 complex formation and decreasing cGMP formation. These studies suggest that hsp90 regulates both eNOS- and sGC-dependent relaxations.
LRP and αvβ3mediate tPA activation of smooth muscle cells
Authors: Akkawi Sa'ed , Nassar Taher , Tarshis Mark , Cines Douglas B. , Higazi Abd Al-Roof
Published: February 2006

Tissue-type plasminogen activator (tPA) regulates vascular contractility through the low-density lipoprotein-related receptor (LRP), and this effect is inhibited by plasminogen activator inhibitor type 1 (PAI-1). We now report that tPA-mediated vasocontraction also requires the integrin αvβ3. tPA-induced contraction of rat aortic rings is inhibited by the Arg-Gly-Asp (RGD) peptide and by monoclonal anti-αvβ3antibody. tPA induces the formation of a complex between LRP and αvβ3in vascular smooth muscle cells. The three proteins are internalized within 10 min, causing the cells to become refractory to the readdition of tPA. LRP and αvβ3return to the cell surface by 90 min, restoring cell responsiveness to tPA. PAI-1 and the PAI-1-derived hexapeptide EEIIMD abolish the vasocontractile activity of tPA and inhibit the tPA-mediated interaction between LRP and αvβ3. tPA induces calcium mobilization from intracellular stores in vascular smooth muscle cells, and this effect is inhibited by PAI-1, RGD, and antibodies to both LRP and αvβ3.These data indicate that tPA-mediated vasocontraction involves the coordinated interaction of LRP with αvβ3. Delineating the mechanism underlying these interactions and the nature of the signals transduced may provide new tools to regulate vascular tone and other consequences of tPA-mediated signaling.
Effects of hypoxia, anoxia, and metabolic inhibitors on KATP channels in rat femoral artery myocytes
Authors: Quayle J. M. , Turner M. R. , Burrell H. E. , Kamishima T.
Published: February 2006

Vascular ATP-sensitive potassium (KATP) channels have an important role in hypoxic vasodilation. Because KATP channel activity depends on intracellular nucleotide concentration, one hypothesis is that hypoxia activates channels by reducing cellular ATP production. However, this has not been rigorously tested. In this study we measured KATP current in response to hypoxia and modulators of cellular metabolism in single smooth muscle cells from the rat femoral artery by using the whole cell patch-clamp technique. KATP current was not activated by exposure of cells to hypoxic solutions (Po2 ∼35 mmHg). In contrast, voltage-dependent calcium current and the depolarization-induced rise in intracellular calcium concentration ([Ca2+]i) was inhibited by hypoxia. Blocking mitochondrial ATP production by using the ATP synthase inhibitor oligomycin B (3 μM) did not activate current. Blocking glycolytic ATP production by using 2-deoxy-d-glucose (5 mM) also did not activate current. The protonophore carbonyl cyanide m-chlorophenylhydrazone (1 μM) depolarized the mitochondrial membrane potential and activated KATP current. This activation was reversed by oligomycin B, suggesting it occurred as a consequence of mitochondrial ATP consumption by ATP synthase working in reverse mode. Finally, anoxia induced by dithionite (0.5 mM) also depolarized the mitochondrial membrane potential and activated KATP current. Our data show that: 1) anoxia but not hypoxia activates KATP current in femoral artery myocytes; and 2) inhibition of cellular energy production is insufficient to activate KATP current and that energy consumption is required for current activation. These results suggest that vascular KATP channels are not activated during hypoxia via changes in cell metabolism. Furthermore, part of the relaxant effect of hypoxia on rat femoral artery may be mediated by changes in [Ca2+]i through modulation of calcium channel activity.
Polyethylene glycol and a novel developed polyethylene glycol-nitric oxide normalize arteriolar response and oxidative stress in ischemia-reperfusion
Authors: Bertuglia S. , Veronese F. M. , Pasut G.
Published: February 2006

Polyethylene glycol (PEG) has been shown to repair cell membranes and, thus, inhibit free radical production in in vitro and in vivo models. We hypothesized that PEG and newly developed organic nitrate forms of PEG (PEG-NO) could repair endothelial dysfunction in ischemia-reperfusion (I/R) injury in the hamster cheek pouch visualized by intravital fluorescent microscopy. After treatments, we evaluated diameter and RBC velocity and flow in arterioles, as well as lipid peroxides in the systemic blood, perfused capillary length, vascular permeability, leukocyte adhesion, and amount of von Willebrand factor (vWF) in the blood after I/R injury. A control group was treated with 5,000- or 10,000-Da PEG, and three groups were treated with PG1 (1 NO molecule covalently bound to PEG, 5,170 Da), PG8 (8 NO molecules covalently bound to PEG, 11,860 Da), and PG16 (16 NO molecules covalently bound to PEG, 14,060 Da). All animals received 0.5 mg/0.5 ml. Lipid peroxides increased at 5 and 15 min of reperfusion, whereas diameter, RBC velocity, and blood flow decreased in arterioles after I/R injury. Vascular permeability, leukocyte adhesion, and vWF increased significantly. PEG and PG1 attenuated lipid peroxides and vasoconstriction during reperfusion and decreased leukocyte adhesion and vascular permeability. PG8 maintained lipid peroxides at normal levels, increased arteriolar diameter, flow, and perfused capillary length, and decreased vWF level and leukocyte adhesion ( P < 0.05). PG16 was less effective than PG1 and PG8. In conclusion, PEG-NO shows promise as a compound that protects microvascular perfusion by normalizing the balance between NO level and excessive production of free radicals in endothelial cells during I/R injury.
Increased cardiac output and microvascular blood flow during mild hemoconcentration in hamster window model
Authors: Martini Judith , Tsai Amy G. , Cabrales Pedro , Johnson Paul C. , Intaglietta Marcos
Published: February 2006

The effect of small hematocrit (Hct) increases on cardiac index (cardiac output/body wt) and oxygen release to the microcirculation was investigated in the awake hamster window chamber model by means of exchange transfusions of homologous packed red blood cells. Increasing Hct between 8 and 13% from baseline increased cardiac index by 5–31% from baseline ( P < 0.05) and significantly lowered systemic blood pressure ( P < 0.05). The relationship between Hct and cardiac index is described by a second-order polynomial ( R2 = 0.84; P < 0.05) showing that Hct increases up to 20% from baseline increase cardiac index, whereas increases over 20% from baseline decrease cardiac index. Combining this data with measurements of blood pressure allowed to determine total peripheral vascular resistance, which was a minimum at 8–13% Hct increase and was described by a second-order polynomial ( R2 = 0.83; P < 0.05). Oxygen measurements in arterioles, venules, and the tissue at 8–13% Hct increase were identical to control; thus, as a consequence of increased flow and oxygen-carrying capacity, oxygen delivery and extraction increased, but the change was not statistically significant. Previous results with the same model showed that the observed effects are related to shear stress-mediated release of nitric oxide, an effect that should be also present in the heart microcirculation, leading to increased blood flow, myocardial oxygen consumption, and contractility. We conclude that a minimum viscosity level is necessary for generating the shear stress required for maintaining normal cardiovascular function.
AMPK activation restores the stimulation of glucose uptake in an in vitro model of insulin-resistant cardiomyocytes via the activation of protein kinase B
Authors: Bertrand Luc , Ginion Audrey , Beauloye Christophe , Hebert Alexandre D. , Guigas Bruno , Hue Louis , Vanoverschelde Jean-Louis
Published: February 2006

Diabetic hearts are known to be more susceptible to ischemic disease. Biguanides, like metformin, are known antidiabetic drugs that lower blood glucose concentrations by decreasing hepatic glucose production and increasing glucose disposal in muscle. Part of these metabolic effects is thought to be mediated by the activation of AMP-activated protein kinase (AMPK). In this work, we studied the relationship between AMPK activation and glucose uptake stimulation by biguanides and oligomycin, another AMPK activator, in both insulin-sensitive and insulin-resistant cardiomyocytes. In insulin-sensitive cardiomyocytes, insulin, biguanides and oligomycin were able to stimulate glucose uptake with the same efficiency. Stimulation of glucose uptake by insulin or biguanides was correlated to protein kinase B (PKB) or AMPK activation, respectively, and were additive. In insulin-resistant cardiomyocytes, where insulin stimulation of glucose uptake was greatly reduced, biguanides or oligomycin, in the absence of insulin, induced a higher stimulation of glucose uptake than that obtained in insulin-sensitive cells. This stimulation was correlated with the activation of both AMPK and PKB and was sensitive to the phosphatidylinositol-3-kinase/PKB pathway inhibitors. Finally, an adenoviral-mediated expression of a constitutively active form of AMPK increased both PKB phosphorylation and glucose uptake in insulin-resistant cardiomyocytes. We concluded that AMPK activators, like biguanides and oligomycin, are able to restore glucose uptake stimulation, in the absence of insulin, in insulin-resistant cardiomyocytes via the additive activation of AMPK and PKB. Our results suggest that AMPK activation could restore normal glucose metabolism in diabetic hearts and could be a potential therapeutic approach to treat insulin resistance.
Functional arrangement of rat diaphragmatic initial lymphatic network
Authors: Grimaldi Annalisa , Moriondo Andrea , Sciacca Laura , Guidali Maria Luisa , Tettamanti Gianluca , Negrini Daniela
Published: February 2006

Fluid and solute flux between the pleural and peritoneal cavities, although never documented under physiological conditions, might play a relevant role in pathological conditions associated with the development of ascitis and pleural effusion and/or in the processes of tumor dissemination. To verify whether a pleuroperitoneal flux might take place through the diaphragmatic lymphatic network, the transdiaphragmatic pressure gradient (ΔPTD) was measured in five spontaneously breathing anesthetized rats. ΔPTD was −1.93 cmH2O (SD 0.59) and −3.1 cmH2O (SD 0.82) at end expiration and at end inspiration, respectively, indicating the existence of a pressure gradient directed from the abdominal to the pleural cavity. Morphometrical analysis of the diaphragmatic lymphatic network was performed in the excised diaphragm of three additional rats euthanized with an anesthesia overdose. Optical and electron microscopy revealed that lymphatic submesothelial lacunae and lymphatic capillaries among the skeletal muscles fibers show the ultrastructural features of the so-called initial lymphatic vessels, namely, a discontinuous basal lamina and anchoring filaments linking the outer surface of the endothelial cells to connective tissue or to muscle fibers. Primary unidirectional valves in the wall of the initial lymphatics allow entrance of serosal fluid into the lymphatic network preventing fluid backflow, while unidirectional intraluminar valves in the transverse vessels convey lymph centripetally toward central collecting ducts. The complexity and anatomical arrangement of the two valves system suggests that, despite the existence of a favorable ΔPTD, in the physiological condition no fluid bulk flow takes place between the pleural and peritoneal cavity through the diaphragmatic lymphatic network.
Brimonidine evokes heterogeneous vasomotor response of retinal arterioles: diminished nitric oxide-mediated vasodilation when size goes small
Authors: Rosa Robert H. , Hein Travis W. , Yuan Zhaoxu , Xu Wenjuan , Pechal Melissa I. , Geraets Ryan L. , Newman Joseph M. , Kuo Lih
Published: February 2006

Brimonidine, an α2-adrenergic receptor (AR) agonist, has been employed in the treatment of glaucoma due to its beneficial effects on intraocular pressure reduction and neuroprotection. In addition, some studies have implicated that brimonidine might influence ocular blood flow; however, its effect on the retinal microcirculation has not been documented. Herein, we examined the vasomotor action of brimonidine on different branching orders of retinal arterioles in vitro and determined the contribution of the α2-AR subtype and the role of endothelium-derived nitric oxide (NO) in this vasomotor response. First- and second-order retinal arterioles of pigs were isolated, cannulated, and pressurized for functional studies. Videomicroscopic techniques were employed to record diameter changes in response to brimonidine. RT-PCR was performed for detection of α-AR and endothelial NO synthase (eNOS) mRNA in retinal arterioles. All first-order arterioles (82 ± 2 μm ID) dilated dose dependently to brimonidine (0.1 nM to 10 μM) with 10% dilation at the highest concentration. Second-order arterioles (50 ± 1 μm ID) responded heterogeneously with either dilation or constriction. The incidence and magnitude of vasoconstriction were increased with increasing brimonidine concentration. Administration of the NO synthase inhibitor NG-nitro-l-arginine methyl ester abolished the brimonidine-induced vasodilation in first- and second-order arterioles. Regardless of vessel size, vasomotor responses (i.e., vasodilation and vasoconstriction) of retinal arterioles were sensitive to the α2-AR antagonist rauwolscine. Consistent with the functional data, α2A-AR and eNOS mRNAs were detected in retinal arterioles. Collectively, our data demonstrate that brimonidine at clinical doses evokes a consistent NO-dependent vasodilation in first-order retinal arterioles but a heterogeneous response in second-order arterioles. These vasomotor responses are mediated by the activation of α2-AR. It appears that brimonidine, depending on the concentration and vessel size, may alter local retinal blood flow.
Pioglitazone prevents cardiac remodeling in high-fat, high-calorie-induced Type 2 diabetes mellitus
Authors: Rodriguez Walter E. , Joshua Irving G. , Falcone Jeff C. , Tyagi Suresh C.
Published: February 2006

The agonists of peroxisome proliferator-activated receptor-γ (PPARγ) ameliorate cardiovascular complications associated with diabetes mellitus. We tested the hypothesis that recovery from ailing to failing myocardium in diabetes by PPARγ agonist is in part due to decreased matrix metalloproteinase-9 (MMP-9) activation and left ventricular (LV) tissue levels of homocysteine (Hcy). C57BL/6J mice were made diabetic (D) by feeding them a high-fat calorie diet. PPARγ was activated by adding pioglitazone (Pi) to the diet. After 6 wk, mice were grouped into: normal calorie diet (N), D, N + Pi and D + Pi ( n = 6 in each group). LV variables were measured by echocardiography, endothelial-myocyte (E-M) coupling was measured in cardiac rings, and MMP-9 activation was measured by zymography. Blood glucose levels were twofold higher in D mice compared with N mice. Pi decreased the levels of glucose in D mice to the levels in N mice. LV Hcy levels were 3.5 ± 0.5 μM in N groups compared with 12.4 ± 0.6 μM in D groups. Treatment with Pi normalized the LV levels of Hcy but had no effect on plasma levels of Hcy. In the D group, LV contraction was reduced compared with that of the N group and was ameliorated by treatment with Pi. LV wall thickness was reduced to 0.25 ± 0.02 mm in the D group compared with 0.42 ± 0.01 mm in the N group. LV diastolic diameter was 3.05 ± 0.01 mm in the D group compared with 2.20 ± 0.02 mm in the N group. LV systolic diameter was 1.19 ± 0.02 mm in the D group and 0.59 ± 0.01 mm in the N group. Pi normalized the LV variables in D mice. The responses to ACh and nitroprusside were attenuated in diabetic hearts, suggesting that there was E-M uncoupling in the D group compared with the N group, which was ameliorated by Pi. Plasma and LV levels of MMP-2 and -9 activities were higher in the D group than in the N group but normalized after Pi treatment. These results suggest that E-M uncoupling in the myocardium, in part, is due to increased MMP activities secondary to suppressing PPARγ activity in high-fat, calorie-induced Type 2 diabetes mellitus.
Analysis of blood flow in an out-of-plane CABG model
Authors: Sankaranarayanan Meena , Ghista Dhanjoo N. , Poh Chua Leok , Seng Tan Yong , Kassab Ghassan S.
Published: February 2006

Coronary artery bypass graft (CABG) is a routine surgical treatment for ischemic and infarcted myocardium. A large number of CABG fail postoperatively because of intimal hyperplasia within months or years. The cause of this failure is thought to be partly related to the flow patterns and shear stresses acting on the endothelial cells. An accurate representation of the flow field and associated wall shear stress (WSS) requires a detailed three-dimensional (3D) model of the CABG. The purpose of this study is to present a detailed analysis of blood flow in a 3D aorto/left CABG, bypassing the occluded left anterior descending coronary (LAD) artery. The analysis takes into account the influence of the out-of-plane geometry of the graft. The finite volume technique was employed to model the 3D blood flow pattern to determine the velocity and WSS distributions. This study presents the flow field distributions of the velocity and WSS at four instances of the cardiac cycle, two in systole and two in diastole. Our results reveal that the CABG geometry has a significant effect on the velocity distribution. The axial velocity profiles at different instances of the cardiac cycle exhibit strong skewing; significant secondary flow and vortex structures are seen in the in-plane velocity patterns. The maximum WSS on the bed of the occluded LAD artery opposite to the graft junction is 14 Pa in middiastole, whereas there is a significantly lower and more uniform distribution of WSS on the bed of the anastomosis. The present results indicate that nonplanarity of the blood vessel along with the inflow conditions has a substantial effect on the fluid mechanics of CABG that contribute to the patency of graft.
Essential role of ROS-mediated NFAT activation in TNF-α induction by crystalline silica exposure
Authors: Ke Qingdong , Li Jingxia , Ding Jin , Ding Min , Wang Liying , Liu Bingci , Costa Max , Huang Chuanshu
Published: February 2006

Occupational exposure to crystalline silica has been associated with progressive pulmonary silicosis and lung cancer, but the underlying molecular mechanisms are not well understood. Previous studies have shown that crystalline silica exposure can generate reactive oxygen species (ROS) and induce the expression of the inflammatory cytokine tumor necrosis factor-α (TNF-α) in cells. TNF-α is believed to be critical in the development of silica-related diseases. Thus it will be of significance to understand the mechanisms of TNF-α induction by silica exposure. Given the fact that the transcription factor nuclear factor of activated T cells (NFAT) plays an important role in the regulation of TNF-α and can also be activated by ROS, in this study we investigated the potential role of ROS in silica-induced NFAT activity as well as TNF-α expression in Cl41 cells. The results showed that exposure of cells to silica led to NFAT transactivation and TNF-α induction, where superoxide anion radical (O2−·), but not H2O2, was involved. The knockdown of NFAT3 by its specific small interfering RNA significantly attenuated the silica-induced TNF-α transcription. This study demonstrated that silica was able to activate NFAT in an O2−·-dependent manner, which was required for TNF-α induction.
Impaired alveolar liquid clearance after 48-h isoproterenol infusion spontaneously recovers by 96 h of continuous infusion
Authors: Maron Michael B. , Folkesson Hans G. , Stader Sonya M. , Hodnichak Cheryl M.
Published: February 2006

We previously demonstrated that 48-h isoproterenol (Iso) infusion in rats impaired the ability of β-adrenoceptor (β-AR) agonists to increase alveolar liquid clearance (ALC). In this study, we determined whether this impairment persisted over longer time periods by infusing 400 μg·kg−1·h−1 Iso by osmotic minipump for 24–144 h ( n = 6–7/group). ALC in control rats was 19.0 ± 2.4 (SD)% of instilled volume absorbed per hour. In Iso-infused rats, ALC was elevated at 24 h (34.9 ± 2.4%) and decreased at 48 h (15.2 ± 4.4%) and had recovered to 24 h values at 96 h (37.3 ± 3.8%) and 144 h (35.2 ± 3.3%). Plasma Iso concentrations remained elevated at all Iso infusion times. Peripheral lung β2-AR expression exhibited a parallel time course, with a reduction in expression observed at 48 h, followed by an increase to 24 h values at 96 and 144 h. Propranolol prevented the increase in ALC observed at 96 and 144 h, indicating that the recovery in ALC was mediated by a recovery of β-AR function and β-AR signaling. ALC at 96 and 144 h could not be further increased by terbutaline, indicating that ALC was maximally stimulated. These data indicate that recovery of β-AR-stimulated ALC can occur in the continued presence of Iso and is mediated by a recovery of the ability of the distal lung epithelium to respond to β-AR stimulation.
Single nucleotide polymorphism in 5′-flanking region reduces transcription of surfactant protein B gene in H441 cells
Authors: Thomas Klaus H. , Meyn Philipp , Suttorp Norbert
Published: February 2006

Surfactant protein (SP)-B is expressed in a cell-specific manner and is essential for surfactant function and survival. Abnormal surfactant function occurs in humans and genetically engineered mice with SP-B levels well below 50% of normal. SP-B mRNA levels vary in fetal lung explants among individuals, possibly due to genetic variety. Polymorphisms within the SP-B gene have been described extensively; however, some of their functional relevance remains unclear. Mutations within the SP-B gene may affect mRNA content, but altered gene transcription or mRNA-stability has not been clearly demonstrated. We characterized a single nucleotide polymorphism (SNP) found in the upstream enhancer of SP-B, consisting of a single base pair change in the consensus sequence of the most downstream-located thyroid transcription factor 1 binding element in the upstream enhancer of the SP-B 5′-flanking region and located at position 384 upstream of the transcriptional start site of the SP-B gene. In a small patient population ( n = 53) we found 70% were homozygous for the wild type (WT), one individual (2%) was homozygous for the polymorphism (Pm), and 28% were heterozygous. To further elucidate possible functions we performed electromobility shift assays with extracts from H441 cells that showed a reduced binding affinity of the mutated sequence compared with WT. In reporter gene assays the Pm caused a reduction of 53% in transcriptional activity compared with WT in transfected H441 cells. Stimulation of these constructs with retinoic acid resulted in enhanced reporter gene activity of both constructs. After stimulation the Pm still exhibited a reduced activity compared with the WT sequence. We conclude that the described SNP causes differences in SP-B transcriptional activity and thus may contribute to individually different SP-B mRNA levels.
Involvement of GATA-3-dependent Th2 lymphocyte activation in airway hyperresponsiveness
Authors: Yamashita Naomi , Tashimo Hiroyuki , Ishida Hirofumi , Matsuo Yukiko , Tamauchi Hidekazu , Terashima Masazumi , Yoshiwara Ikuo , Habu Sonoko , Ohta Ken
Published: February 2006

The pathophysiological characteristics of bronchial asthma consist of chronic inflammation of airways, airway hyperresponsiveness, and bronchoconstriction. Studies have shown that T helper type 2 (Th2) cytokines produced by both T cells and mast cells in the airway contribute substantially to the initiation of inflammation in both experimental and human bronchial asthma. GATA-3 is a transcription factor essential to the production of Th2 cytokines by T lymphocytes. To clarify the role of GATA-3-expressing T cells in the pathophysiology of bronchial asthma, we utilized transgenic (Tg) mice carrying the GATA-3 gene and the ovalbumin (OVA)-specific T cell receptor gene (GATA-3-Tg/OVA-Tg). Mice were intranasally administrated OVA without systemic immunization. Airway responses were analyzed with noninvasive and invasive whole body plethysmographs. GATA-3-Tg/OVA-Tg mice exhibited significantly higher IL-13 and IL-4 protein expression in the airway. Although there were no differences in the types of infiltrating cells between GATA-3-Tg/OVA-Tg and GATA-3-non-Tg/OVA-Tg mice and no significant increase in IgE level in either group compared with nontreated mice, the response after ACh inhalation was significantly elevated in GATA-3-Tg/OVA-Tg on the seventh day of intranasal treatment with OVA. This hyperresponsiveness was inhibited by 5-lipoxygenase inhibitor and IL-13 neutralization, suggesting that airway responses were induced through IL-13 and leukotriene pathway. In conclusion, airway hyperresponsiveness, a characteristic of bronchial asthma, is regulated at the level of GATA-3 transcription by T lymphocytes in vivo.
Increased secretion of leukemia inhibitory factor by immature airway smooth muscle cells enhances intracellular signaling and airway contractility
Authors: Fayon Michael , Rebola Muriel , Berger Patrick , Daburon Sophie , Ousova Olga , Lavrand Frédéric , Moukaïla Boutchi , Pujol Wilfried , Taupin Jean Luc , Labbé André , Molimard Mathieu , Marthan Roger
Published: February 2006

Airway smooth muscle cells (ASMC) play a major role in airway inflammation, hyperresponsiveness, and obstruction in asthma. However, very little is known regarding the relation between inflammatory mediators and cytokines and immature ASMC. The aim of this study was to evaluate 1) the secretion of leukemia inhibitory factor (LIF) (an IL-6 family neurotrophic cytokine) by ASMC; 2) intracellular calcium concentration ([Ca2+]i) signaling; and 3) the effect of LIF on mast cell chemotaxis and rat airway contractility. Immature and adult human ASMC were cultured. ELISA and real-time PCR were performed to assess LIF protein secretion and mRNA production, [ methyl-3H]thymidine incorporation to quantify ASMC DNA synthesis, a Boyden chamber to evaluate the effect of LIF on mast cell chemotaxis, microspectroflurimetry using indo-1 (at baseline and after stimulation bradykinin, U-46619, histamine, and acetylcholine, in the presence or absence of LIF or TNF-α) for [Ca2+]i signaling, and isolated rat pup tracheae to determine the effect of LIF on airway contractility to ACh. TNF-α-stimulated immature ASMC produce more LIF mRNA and protein than adult ASMC, although this cytokine induces a moderate increase in DNA synthesis (+20%) in adult ASMC only. Human recombinant LIF exerts no chemotactic effect on human mast cells. In immature ASMC, ACh-induced [Ca2+]i response was enhanced twofold after incubation with LIF, whereas TNF-α increased the [Ca2+]i to U-46619 threefold. In TNF-α-exposed adult ASMC, [Ca2+]i responses to ACh were of greater magnitude (sixfold increase) than in immature ASMC. Human recombinant LIF increased contractility to ACh by 50% in immature, isolated rat tracheae. Stimulated immature human ASMC greatly secrete LIF, thus potentially contributing to neuroimmune airway inflammation and subsequent remodeling. Increased LIF secretion enhances airway reactivity and [Ca2+]i signaling.
Differentiation of human airway epithelia is dependent on erbB2
Authors: Vermeer Paola D. , Panko Lacey , Karp Philip , Lee John H. , Zabner Joseph
Published: February 2006

A clinical case documented a reversible change in airway epithelial differentiation that coincided with the initiation and discontinuation of trastuzumab, an anti-erbB2 antibody. This prompted the investigation into whether blocking the erbB2 receptor alters differentiation of the airway epithelium. To test this hypothesis, we treated an in vitro model of well-differentiated human airway epithelia with trastuzumab or heregulin-α, an erbB ligand. In addition, coculturing with human lung fibroblasts tested whether in vivo subepithelial fibroblasts function as an endogenous source of ligands able to activate erbB receptors expressed by the overlying epithelial cells. Epithelia were stained with hematoxylin and eosin and used for morphometric analysis. Trastuzumab treatment decreased the ciliated cell number by 49% and increased the metaplastic, flat cell number by 640%. Heregulin-α treatment increased epithelial height and decreased the number of metaplastic and nonciliated columnar cells, whereas it increased the goblet cell number. We found that normal human lung fibroblasts express transforming growth factor-α, heparin-binding epidermal-like growth factor, epiregulin, heregulin-α, and amphiregulin, all of which are erbB ligands. Cocultures of airway epithelia with primary fibroblasts increased epithelial height comparable to that achieved following heregulin-α treatment. These data show that erbB2 stimulation is required for maintaining epithelial differentiation. Furthermore, the mesenchyme underlying the airway epithelium secretes a variety of erbB ligands that may direct various pathways of epithelial differentiation.
Effects of arterial oxygen content on peripheral locomotor muscle fatigue
Authors: Amann Markus , Romer Lee M. , Pegelow David F. , Jacques Anthony J. , Hess C. Joel , Dempsey Jerome A.
Published: February 2006

The effect of arterial O2content (CaO2) on quadriceps fatigue was assessed in healthy, trained male athletes. On separate days, eight participants completed three constant-workload trials on a bicycle ergometer at fixed workloads (314 ± 13 W). The first trial was performed while the subjects breathed a hypoxic gas mixture [inspired O2fraction (FiO2) = 0.15, Hb saturation = 81.6%, CaO2= 18.2 ml O2/dl blood; Hypo] until exhaustion (4.5 ± 0.4 min). The remaining two trials were randomized and time matched with Hypo. The second and third trials were performed while the subjects breathed a normoxic (FiO2= 0.21, Hb saturation = 95.0%, CaO2= 21.3 ml O2/dl blood; Norm) and a hyperoxic (FiO2= 1.0, Hb saturation = 100%, CaO2= 23.8 ml O2/dl blood; Hyper) gas mixture, respectively. Quadriceps muscle fatigue was assessed via magnetic femoral nerve stimulation (1–100 Hz) before and 2.5 min after exercise. Myoelectrical activity of the vastus lateralis was obtained from surface electrodes throughout exercise. Immediately after exercise, the mean force response across 1–100 Hz decreased from preexercise values ( P < 0.01) by −26 ± 2, −17 ± 2, and −13 ± 2% for Hypo, Norm, and Hyper, respectively; each of the decrements differed significantly ( P < 0.05). Integrated electromyogram increased significantly throughout exercise ( P < 0.01) by 23 ± 3, 10 ± 1, and 6 ± 1% for Hypo, Norm, and Hyper, respectively; each of the increments differed significantly ( P < 0.05). Mean power frequency fell more ( P < 0.05) during Hypo (−15 ± 2%); the difference between Norm (−7 ± 1%) and Hyper (−6 ± 1%) was not significant ( P = 0.32). We conclude that ΔCaO2during strenuous systemic exercise at equal workloads and durations affects the rate of locomotor muscle fatigue development.
Atrial Natriuretic Peptide Predicts Impaired Atrial Remodeling and Occurrence of Late Postoperative Atrial Fibrillation after Surgery for Symptomatic Aortic Stenosis
Authors: Yilmaz Mehmet Birhan , Erbay Ali Riza , Balci Mustafa , Guray Yesim , Cihan Gokhan , Guray Umit , Kisacik Halil L. , Korkmaz Sule
Published: February 2006

Background: Aortic stenosis (AS) and atrial fibrillation (AF) are commonly encountered in clinical practice. Natriuretic peptides (NP) are endogenous cardiac hormones, which have been shown to increase in patients with heart failure, and valvular or congenital heart disease. We aimed to determine the association between atrial NP (ANP) and late postoperative AF after surgery for AS along with temporal changes in plasma ANP levels and left atrial (LA) volumes. Methods: 22 patients (16 males/6 females, mean age: 61 years) with symptomatic AS and 8 healthy volunteers (5 males/3 females) were enrolled into our study. All the patients studied underwent transthoracic echocardiography, which was repeated during the follow-up. N-terminal ANP (N-ANP) was studied initially and at the 2-month follow-up. Postoperatively, the patients were followed up for 12 months for AF attacks. Results: Patients with AS had significantly higher levels of N-ANP, left ventricular (LV) end-diastolic pressure, E/A ratio, LV mass and LA volumes compared to the controls. Patients with postoperative AF attacks were significantly older, had higher N-ANP levels and LV end-diastolic pressure in addition to higher LA volumes and longer symptom duration compared to patients without AF. Age at the time of operation (p = 0.011) and N-ANP at the 2nd month (p = 0.047) were found to be independent predictors for late AF attacks during follow-up in regression analysis. Besides, N-ANP (p < 0.001) at the 2-month follow-up independently predicted impaired LA remodeling. Conclusion: ANP might be an important factor to identify AS patients at risk for late postoperative AF attacks.
Consistent Expression of HGF and c-met in the Perinatal Lung
Authors: Lassus P. , Janer J. , Haglund C. , Karikoski R. , Andersson L.C. , Andersson S.
Published: February 2006

Background: Hepatocyte growth factor (HGF), an epithelial cell mitogen, has been shown to participate in normal lung development and in regeneration after lung injury. In human preterm infants, lower pulmonary HGF has been associated with more severe respiratory disease. Objectives: We studied the protein expression of HGF and its receptor c-met during the perinatal period in the human lung. Methods: Immunohistochemistry for HGF and c-met was performed on lung tissues from autopsies of 4 fetuses, 5 preterm infants, 5 term infants, and 4 infants with bronchopulmonary dysplasia. Results: Immunohistochemistry for HGF showed staining in all cases in mesenchymal cells (fibroblasts and cartilage cells). Additional staining was found in bronchial and distal airway epithelium. Immunohistochemistry for c-met showed staining in bronchial and distal airway epithelium, and in most cases in neutrophils. Conclusions: The consistent expression of HGF and c-met during the perinatal period supports a physiological role for HGF in human lung development.
Iodine Saturation of Roma Neonates in Prague Is Not at an Optimum Level
Authors: Dlouhý P. , Rambousková J. , Wiererová O. , Pokorný R. , Bílek R. , Kubisová D. , Procházka B. , Anděl M.
Published: February 2006

Aim: The purpose of our study was to determine urinary iodine as an indicator of iodine supplementation in Roma (Gypsy) neonates compared to majority population neonates. Methods: The groups studied were formed by 30 full-term Roma neonates and 151 majority population neonates. Iodine was determined from samples of urine collected on the 4th day after delivery, after alkaline ashing, using the Sandell-Kolthoff method. Results: The median of urinary iodine in Roma neonates was 92.15 µg/l urine and in neonates from majority population mothers it was 109.20 µg/l urine. The mean of urinary iodine in Roma neonates was 114.55 µg/l urine (SD 71.68 µg/l) and in neonates from majority population mothers it was 141.86 µg/l urine (SD 87.42 µg/l). The difference was not statistically significant. Majority population mothers more frequently consumed nutrition supplements containing iodine as well as fish. Conclusions: Compared to older data, supplementation of neonates with iodine is higher. However, it does not reach optimum levels. The urinary iodine median in Roma neonates lies in the mild iodine deficiency band.
Event-Related Visual versus Blocked Motor Task: Detection of Specific Cortical Activation Patterns with Functional Near-Infrared Spectroscopy
Authors: Plichta M.M. , Herrmann M.J. , Ehlis A.-C. , Baehne C.G. , Richter M.M. , Fallgatter A.J.
Published: February 2006

The purpose of this study was to investigate the regional specificity of multi-channel functional near-infrared spectroscopy (fNIRS) in the detection of cortical activation in humans. Therefore, brain activation evoked by a visual as well as a motor task was examined using 52-channel fNIRS. Analyses demonstrated an isolated activation in the occipital area during visual stimulation, whereas other regions exhibited little or no activation. Analyses of the motor task data clearly identified a differential activation pattern. The observation of an extensive cortical area by multi-channel measurement during two different tasks made it possible to examine the extent to which fNIRS measurements detect regional specific activations. We conclude that fNIRS measurements can detect regionally isolated cortical activation.
Histopathologic Examination for Helicobacter pylori as a Possible Etiopathogenic Factor in Laryngeal Carcinoma
Authors: Kizilay Ahmet , Saydam Levent , Aydin Abdullah , Kalcioglu M. Tayyar , Ozturan Orhan , Aydin N. Engin
Published: February 2006

Objective: We investigated the presence of Helicobacter pylori in laryngeal cancer specimens to reveal whether or not this is a risk factor in the development of squamous cell carcinoma of the larynx. Methods: Sixty-nine total laryngectomy specimens with the pathologic diagnosis of squamous cell carcinoma and 30 laryngeal tissue samples that had been taken for the investigation of nonneoplastic (polyp, nodule) diseases were studied. Specimens of both tumor and control groups were stained with hematoxylin-eosin and modified Giemsa stains, and then they were examined under a light microscope. Results: In both groups, H. pylori could not be found in any of the cases. Conclusion: The histologic examination of our series did not reveal any clue related to the possible etiologic association between H. pylori and squamous cell carcinoma of the larynx.
In vivo Evaluation of the Genotoxic Effects of Clomiphene Citrate on Rat Reticulocytes: A Micronucleus Genotoxicity
Authors: Duran Bulent , Ozdemir Ismail , Demirel Yeltekin , Ozdemir Ozturk , Cetin Ali , Guven Aysel
Published: February 2006

Objective: To determine the genotoxic effects of clomiphene citrate (CC) on rat reticulocytesin vivo. Methods: In this prospective, randomized, controlled study, rats were each assigned randomly to the CC 50, CC 100, CC 200, or control group and were given repeat doses of 0.16, 0.32 or 0.64 mg CC, or normal saline, respectively. Each study group received its CC dose in 2 ml of saline intraperitoneally for 5 days, while the control group received only 2 ml of saline. Each treatment cycle was repeated six times. Six months later, the rats were euthanized. Bone marrow tissues were removed, and pluripotent reticulocyte cells with micronuclei, nuclear buds, and binuclear abnormalities were analyzed using an in situmicronuclei assay under light microscopy. The proportion of micronucleated erythrocytes was measured. Results: Fewer cells with nuclear buds and binuclear abnormalities were detected in the CC 50 group and controls. The CC 100 and 200 groups had significantly (p < 0.05) more nuclear buds and binuclear abnormalities compared with the CC 50 group and controls in the cytogenetic analysis of bone marrow stem cells. Conclusion: In rats, the micronucleus genotoxicity assay suggests a dose-dependent CC effect on genomic instability in bone marrow stem cells in vivo.
Competitive Binding of CREB and ATF2 to cAMP/ATF Responsive Element Regulates eNOS Gene Expression in Endothelial Cells
Authors: Niwano Kazuo , Arai Masashi , Koitabashi Norimichi , Hara Shiro , Watanabe Atai , Sekiguchi Kenichi , Tanaka Toru , Iso Tatsuya , Kurabayashi Masahiko
Published: February 2006

Objective—

Expression of endothelial nitric oxide synthase (eNOS) is a critical determinant for vascular homeostasis. We examined the effects of Beraprost sodium (BPS), a stable analogue of prostacyclin, on the eNOS gene expression in the presence of inflammatory cytokine interleukin (IL)-1β in cultured endothelial cells.

Method and Results—

Exposure of human and bovine endothelial cells to IL-1β decreased eNOS expression. Western blot analysis using phospho-specific antibodies showed that IL-1β stimulated p38 MAP kinase and phosphorylated ATF2. BPS inhibited these effects via protein kinase A (PKA)/cAMP-responsive element binding protein (CREB) activation. Transfection assays using site-specific mutation constructs showed that CRE/ATF elements located at −733 and −603 within the human eNOS promoter are necessary for full IL-1β responsiveness. BPS attenuated the IL-1β–mediated decrease in eNOS promoter activity and the expression of eNOS gene through PKA pathway. Electrophoretic gel mobility shift assays showed that IL-1β increased the binding of phosphorylated ATF2 to CRE/ATF. On treatment with BPS, phosphorylated CREB predominantly bound to CRE/ATF.

Conclusions—

These results indicate that IL-1β and BPS antagonistically regulates the eNOS expression through the activation of p38 and PKA, respectively. Furthermore, the ability to bind both CREB and ATF2 implicates the CRE/ATF sequence as a potential target for multiple signaling pathways in the regulation of the eNOS gene transcription.