bluuebunny/crossref_metadata_2025 huggingface dataset:
Decreased development of necrotizing enterocolitis in IL-18-deficient mice
Authors: Halpern Melissa D. , Khailova Ludmila , Molla-Hosseini Dania , Arganbright Kelly , Reynolds Charity , Yajima Masako , Hoshiba Junji , Dvorak Bohuslav
Published: October 2007

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease predominantly of prematurely born infants, characterized in its severest from by extensive hemorrhagic inflammatory necrosis of the distal ileum and proximal colon. Proinflammatory cytokines have been implicated in the development of NEC, and we have previously shown that IL-18 is significantly elevated in the well-established neonatal rat model of NEC. To determine whether IL-18 contributes to intestinal pathology in NEC, we subjected IL-18 knockout mice to the protocol used to develop experimental NEC in newborn rats. Newborn B6.129P2- Il18tm1Aki/J (NEC IL-18−/−) and wild-type (NEC WT) mice were hand fed every 3 h with cow's milk-based formula and exposed to asphyxia and cold stress twice daily. After 72 h, animals were killed and distal ileum and liver were removed. Disease development was determined via histological changes in the ileum as scored by a blinded evaluator. The number of TNF-α-, IL-12-, and IL-1β-positive cells and macrophages were determined in both ileum and liver via immunohistology. IκB-α and IκB-β were determined from protein extracts from both ileum and liver using Western blot analysis. The incidence and severity of NEC was significantly reduced in NEC IL-18−/−mice compared with NEC WT. Furthermore, mean ileal macrophages and hepatic IL-1β were significantly reduced in IL-18−/−mice subjected to the NEC protocol. There were no statistically significant changes in Kupffer cells, hepatic TNF-α, ileal IL-1β, or IL-12. IκB-α and IκB-β were significantly increased in NEC IL-18−/−mice ileum and liver, respectively. These results confirm that IL-18 plays a crucial role in experimental NEC pathogenesis.
A new model for nonalcoholic steatohepatitis in the rat utilizing total enteral nutrition to overfeed a high-polyunsaturated fat diet
Authors: Baumgardner January N. , Shankar Kartik , Hennings Leah , Badger Thomas M. , Ronis Martin J. J.
Published: October 2007

We have used total enteral nutrition (TEN) to moderately overfeed rats high-polyunsaturated fat diets to develop a model for nonalcoholic steatohepatitis (NASH). Male Sprague-Dawley rats were fed by TEN a 187 kcal·kg−3/4·day−1diet containing 5% (total calories) corn oil or a 220 kcal·kg−3/4·day−1diet in which corn oil constituted 5, 10, 25, 35, 40, or 70% of total calories for 21 or 65 days. Rats fed the 5% corn oil, 220 kcal·kg−3/4·day−1diet had greater body weight gain ( P ≤ 0.05), fat mass ( P ≤ 0.05), and serum leptin and glucose levels ( P ≤ 0.05), but no liver pathology. A dose-dependent increase in hepatic triglyceride deposition occurred with increase in percent corn oil in the 220 kcal·kg−3/4·day−1groups ( P ≤ 0.05). Steatosis, macrophage infiltration, apoptosis, and focal necrosis were present in the 70% corn oil group, accompanied by elevated serum alanine aminotransferase (ALT) levels ( P ≤ 0.05). An increase in oxidative stress (thiobarbituric acid-reactive substances) and TNF-α expression ( P ≤ 0.05) was observed in the 70% corn oil group, as well as an increase in hepatic CYP2E1 and CYP4A1 expression ( P ≤ 0.05). Significant positive correlations were observed between the level of dietary corn oil and the degree of pathology, ALTs, oxidative stress, and inflammation. Liver pathology was progressive with increased necrosis, accompanied by fibrosis, observed after 65 days of TEN. Increased expression of CD36 and l-fabp mRNA suggested development of steatosis was associated with increased fatty acid transport. These data suggest that intragastric infusion of a high-polyunsaturated fat diet at a caloric level of 17% excess total calories results in pathology similar to clinical NASH.
eNOS involved in colitis-induced mucosal blood flow increase
Authors: Petersson Joel , Schreiber Olof , Steege Andreas , Patzak Andreas , Hellsten Anna , Phillipson Mia , Holm Lena
Published: October 2007

The role of NO in inflammatory bowel disease is controversial. Studies indicate that endothelial nitric oxide synthase (eNOS) might be involved in protecting the mucosa against colonic inflammation. The aim of this study was to investigate the involvement of nitric oxide (NO) in regulating colonic mucosal blood flow in two different colitis models in rats. In anesthetized control and colitic rats, the distal colon was exteriorized and the mucosa visualized. Blood flow (laser-Doppler flowmetry) and arterial blood pressure were continuously monitored throughout the experiments, and vascular resistance was calculated. Trinitrobenzene sulfonic acid (TNBS) or dextran sulfate sodium (DSS) was used to induce colitis. All groups were given the NOS inhibitor Nω-nitro-l-arginine (l-NNA) or the inducible NOS (iNOS) inhibitor l- N6-(1-iminoethyl)-lysine (l-NIL). iNOS, eNOS, and neuronal NOS (nNOS) mRNA in colonic samples were investigated with real-time RT-PCR. Before NOS inhibition, colonic mucosal blood flow, expressed as perfusion units, was higher in both colitis models compared with the controls. The blood flow was reduced in the TNBS- and DSS-treated rats during l-NNA administration but was not altered in the control group. Vascular resistance increased more in the TNBS- and DSS-treated rats than in the control rats, indicating a higher level of vasodilating NO in the colitis models. l-NIL did not alter blood pressure or blood flow in any of the groups. iNOS and eNOS mRNA increased in both colitis models, whereas nNOS remained at the control level. TNBS- and DSS-induced colitis results in increased colonic mucosal blood flow, most probably due to increased eNOS activity.
Phenotypic differences in PFIC2 and BRIC2 correlate with protein stability of mutant Bsep and impaired taurocholate secretion in MDCK II cells
Authors: Kagawa Tatehiro , Watanabe Norihito , Mochizuki Kaori , Numari Asano , Ikeno Yoshie , Itoh Johbu , Tanaka Hirotoshi , Arias Irwin M. , Mine Tetsuya
Published: October 2007

Progressive familial cholestasis (PFIC) 2 and benign recurrent intrahepatic cholestasis (BRIC) 2 are caused by mutations in the bile salt export pump (BSEP, ABCB11) gene; however, their prognosis differs. PFIC2 progresses to cirrhosis and requires liver transplantation, whereas BRIC2 is clinically benign. To identify the molecular mechanism(s) responsible for the phenotypic differences, eight PFIC2 and two BRIC2 mutations were introduced in rat Bsep, which was transfected in MDCK II cells. Taurocholate transport activity, protein expression, and subcellular distribution of these mutant proteins were studied in a polarized MDCK II monolayer. The taurocholate transport activity was approximately half of the wild-type (WT) in BRIC2 mutants (A570T and R1050C), was substantially less in two PFIC2 mutants (D482G and E297G), and was almost abolished in six other PFIC2 mutants (K461E, G982R, R1153C, R1268Q, 3767–3768insC, and R1057X). Bsep protein expression levels correlated closely with transport activity, except for R1057X. The half-life of the D482G mutant was shorter than that of the WT (1.35 h vs. 3.49 h in the mature form). BRIC2 mutants and three PFIC mutants (D482G, E297G, and R1057X) were predominantly distributed in the apical membrane. The other PFIC2 mutants remained intracellular. The R1057X mutant protein was stably expressed and trafficked to the apical membrane, suggesting that the COOH-terminal tail is required for transport activity but not for correct targeting. In conclusion, taurocholate transport function was impaired in proportion to rapid degradation of Bsep protein in the mutants, which were aligned in the following order: A570T and R1050C > D482G > E297G > K461E, G982R, R1153C, R1268Q, 3767–3768insC, and R1057X. These results may explain the phenotypic difference between BRIC2 and PFIC2.
Intestinal alkaline phosphatase: selective endocytosis from the enterocyte brush border during fat absorption
Authors: Hansen Gert H. , Niels-Christiansen Lise-Lotte , Immerdal Lissi , Nystrøm Birthe T. , Danielsen E. Michael
Published: October 2007

Absorption of dietary fat in the small intestine is accompanied by a rise of intestinal alkaline phosphatase (IAP) in the serum and of secretion of IAP-containing surfactant-like particles from the enterocytes. In the present work, fat absorption was studied in organ cultured mouse intestinal explants. By immunofluorescence microscopy, fat absorption caused a translocation of IAP from the enterocyte brush border to the interior of the cell, whereas other brush-border enzymes were unaffected. By electron microscopy, the translocation occurred by a rapid (5 min) induction of endocytosis via clathrin-coated pits. By 60 min, IAP was seen in subapical endosomes and along membranes surrounding fat droplets. IAP is a well-known lipid raft-associated protein, and fat absorption was accompanied by a marked change in the density and morphology of the detergent-resistant membranes harboring IAP. A lipid analysis revealed that fat absorption caused a marked increase in the microvillar membrane contents of free fatty acids. In conclusion, fat absorption rapidly induces a transient clathrin-dependent endocytosis via coated pits from the enterocyte brush border. The process selectively internalizes IAP and may contribute to the appearance of the enzyme in serum and surfactant-like particles.
Postinfarct sympathetic hyperactivity differentially stimulates expression of tyrosine hydroxylase and norepinephrine transporter
Authors: Parrish Diana C. , Gritman Kurt , Van Winkle Donna M. , Woodward William R. , Bader Michael , Habecker Beth A.
Published: October 2007

The balance between norepinephrine (NE) synthesis, release, and reuptake is disrupted after acute myocardial infarction, resulting in elevated extracellular NE. Stimulation of sympathetic neurons in vitro increases NE synthesis and the synthetic enzyme tyrosine hydroxylase (TH) to a greater extent than it increases NE reuptake and the NE transporter (NET), which removes NE from the extracellular space. We used TGR(ASrAOGEN) transgenic rats, which lack postinfarct sympathetic hyperactivity, to test the hypothesis that increased cardiac sympathetic nerve activity accounts for the imbalance in TH and NET expression in these neurons after myocardial infarction. TH and NET mRNA levels were identical in the stellate ganglia of unoperated TGR(ASrAOGEN) rats compared with Sprague Dawley (SD) controls, but the threefold increase in TH and twofold increase in NET mRNA seen in the stellate ganglia of SD rats 1 wk after ischemia-reperfusion was absent in TGR(ASrAOGEN) rats. Similarly, the increase in TH and NET protein observed in the base of the SD ventricle was absent in the base of the TGR (ASrAOGEN) ventricle. Neuronal TH content was depleted in the left ventricle of both genotypes, whereas NET was unchanged. Basal heart rate and cardiac function were similar in both genotypes, but TGR(ASrAOGEN) hearts were more sensitive to the β-agonist dobutamine. Tyramine-induced release of endogenous NE generated similar changes in ventricular pressure and contractility in both genotypes, but postinfarct relaxation was enhanced in TGR(ASrAOGEN) hearts. These data support the hypothesis that postinfarct sympathetic hyperactivity is the major stimulus increasing TH and NET expression in cardiac neurons.
Theoretical and electrophysiological evidence for axial loading about aortic baroreceptor nerve terminals in rats
Authors: Feng Bin , Li Bai-yan , Nauman Eric A. , Schild John H.
Published: October 2007

Arterial baroreceptors are essential for neurocirculatory control, providing rapid hemodynamic feedback to the central nervous system. The pressure-dependent discharge of carotid and aortic baroreceptor afferents has been extensively studied. A common assumption has been that circumferential deformation of the arterial wall is the predominant mechanical force affecting baroreceptor discharge. However, in vivo the arterial tree is under significant longitudinal tension, leading to the hypothesis that axially directed forces may contribute to baroreceptor function. To test this hypothesis, we utilized a combination of finite element modeling methods and an in vitro rat aortic arch preparation. Model formulation utilized traditional analytic constructs available in the literature followed by refinement of model material parameters through direct comparison of computationally and experimentally generated pressure-diameter curves. The numerical simulations strongly indicated a functional role for axial loading within the region of the baroreceptive nerve terminal. This prediction was confirmed through single-fiber recording of baroreceptor nerve discharge under conditions with and without longitudinal tension in the vessel preparation. The recordings ( n = 5) demonstrated that longitudinal tension significantly ( P < 0.02) lowered both the pressure threshold (Pth, mmHg) for baroreceptor discharge and sensitivity (Sth, Hz/mmHg). The effect was nearly instantaneous and sustained; i.e., under longitudinal tension average Pth was 84 ± 3 mmHg and Sth was 0.71 ± 0.15 Hz/mmHg, which immediately increased to a Pth of 94 ± 4 mmHg and a Sth of 1.20 ± 0.32 Hz/mmHg with loss of axial tension. Possible explanations of how an abrupt change in axial loading could result in a synchronized increase in afferent drive of the baroreceptor reflex, and the potentiating effect this could have on neurogenically mediated orthostatic intolerance are discussed.
Microvascular transport model predicts oxygenation changes in the infarcted heart after treatment
Authors: Wang Bin , Scott Robert C. , Pattillo Christopher B. , Prabhakarpandian Balabhaskar , Sundaram Shankar , Kiani Mohammad F.
Published: October 2007

Chronic heart failure is most commonly due to ischemic cardiomyopathy after a previous myocardial infarction (MI). Rebuilding lost myocardium to prevent heart failure mandates a neovasculature able to nourish new cardiomyocytes. Previously we have used a series of novel techniques to directly measure the ability of the scar neovasculature to deliver and exchange oxygen at 1–4 wk after MI in rats following left coronary artery ligation. In this study, we have developed a morphologically realistic mathematical model of oxygen transport in cardiac tissue to help in deciding what angiogenic strategies should be used to rebuild the vasculature. The model utilizes microvascular morphology of cardiac tissue based on available morphometric images and is used to simulate experimentally measured oxygen levels after MI. Model simulations of relative oxygenation match experimental measurements closely and can be used to simulate distributions of oxygen concentration in normal and infarcted rat hearts. Our findings indicate that both vascular density and vascular spatial distribution play important roles in cardiac tissue oxygenation after MI. Furthermore, the model can simulate relative changes in tissue oxygen levels in infarcted tissue treated with proangiogenic compounds such as losartan. From the minimum oxygen concentration myocytes need to maintain their normal function, we estimate that 2 wk after MI 29% of the myocardium is severely hypoxic and that the vascular density of the infarcted tissue should reach 75% of normal tissue to ensure that no areas of the myocardium are critically hypoxic.
Vasorelaxant action of 17β-estradiol in rat uterine arteries: role of nitric oxide synthases and estrogen receptors
Authors: Scott Pierre-André , Tremblay André , Brochu Michèle , St-Louis Jean
Published: October 2007

The uterine vasculature plays an important role during pregnancy by providing adequate perfusion of the maternal-fetal interface. To this end, substantial remodeling of the uterine vasculature occurs with consequent changes in responsiveness to contractile agents. The purpose of our study was to characterize the vasorelaxant effects of estrogens on vascular smooth muscles of the rat uterine artery during pregnancy and to evaluate the involvement of estrogen receptors (ESR) and nitric oxide synthases (NOS). To do so, we measured NOS expression in the whole uterine and mesenteric circulatory bed by Western blotting. Vasorelaxant effects of 17β-estradiol (17β-E2) were assessed on endothelium-denuded uterine arteries with wire myographs in the absence and presence of pharmacological modulators [nitro-l-arginine methyl ester (l-NAME), ICI-182780, tamoxifen]. All experiments were performed on arteries from nonpregnant (NP) and late pregnant (P) rats. In the uterine vasculature of the latter group, NOS3 (endothelial NOS) expression was increased, while NOS1 (neuronal NOS) was reduced compared with NP rats. Expression of the NOS2 (inducible NOS) isoform was undetectable in the two groups. Both 17β-E2 and 17α-E2 induced uterine artery relaxation, but the latter evoked lower responses. Endothelium-denuded arteries from NP rats showed larger relaxation with 17β-E2 than P rats. This larger relaxation disappeared in the presence of l-NAME. The ESR antagonist ICI-182780 did not affect acute relaxation with 17β-E2 and 17α-E2. Moreover, membrane-nonpermeant 17β-E2:BSA (estradiol conjugated to bovine serum albumin) did not induce any vasorelaxation. Our results indicate that estrogens exert direct acute vasorelaxant effects in smooth muscles of the rat uterine artery that are mediated by mechanisms independent of ESR activation, but with some stereospecificity. Part of this effect, in NP rats only, is due to nitric oxide produced from muscle NOS1.
Identification of a PKCε-dependent regulation of myocardial contraction by epicatechin-3-gallate
Authors: Li Daxiang , Yang Changjun , Chen Ying , Tian Jiang , Liu Lijun , Dai Qiuping , Wan Xiaochun , Xie Zijian
Published: October 2007

In this study, the effects of tea catechins and tea theaflavins on myocardial contraction were examined in isolated rat hearts using a Langendorff-perfusion system. We found that both tea catechins and theaflavins had positive inotropic effects on the myocardium. Of the tested chemicals, epicatechin-3-gallate (ECG) and theaflavin-3,3′-digallate (TF4) appear to be the most effective tea catechin and theaflavin, respectively. Further studies of ECG-induced positive inotropy revealed the following insights. First, unlike digitalis drugs, ECG had no effect on intracellular Ca2+ level in cultured adult cardiac myocytes. Second, it activated PKCε, but not PKCα, in the isolated hearts as well as in cultured cells. Neither a phospholipase C (PLC) inhibitor ( U73122) nor the antioxidant N-acetyl cysteine (NAC) affected the ECG-induced activation of PKCε. Third, inhibition of PKCε by either chelerythrine chloride (CHE) or PKCε translocation inhibitor peptide (TIP) caused a partial reduction of ECG-induced increases in myocardial contraction. Moreover, NAC was also effective in reducing the effects of ECG on myocardial contraction. Finally, pretreatment of the heart with both CHE and NAC completely abolished ECG-induced inotropic effects on the heart. Together, these findings indicate that ECG can regulate myocardial contractility via a novel PKCε-dependent signaling pathway.
Microvascular oxygen tension in the rat mesentery
Authors: Golub Aleksander S. , Barker Matthew C. , Pittman Roland N.
Published: October 2007

Longitudinal Po2profiles in the microvasculature of the rat mesentery were studied using a novel phosphorescence quenching microscopy technique that minimizes the accumulated photoconsumption of oxygen by the method. Intravascular oxygen tension (Po2, in mmHg) and vessel diameter ( d, in μm) were measured in mesenteric microvessels ( n = 204) of seven anesthetized rats (275 g). The excitation parameters were as follows: 7 × 7-μm spot size; 410 nm laser; and 100 curves at 11 pulses/s, with pulse parameters of 2-μs duration and 80-pJ/μm2energy density. The mean Po2(± SE) was 65.0 ± 1.4 mmHg ( n = 78) for arterioles ( d = 18.8 ± 0.7 μm), 62.1 ± 2.0 mmHg ( n = 38) at the arteriolar end of capillaries ( d = 7.8 ± 0.3 μm), and 52.0 ± 1.0 mmHg ( n = 88) for venules ( d = 22.5 ± 1.0 μm). There was no apparent dependence of Po2on d in arterioles and venules. There were also no significant deviations in Po2based on d (bin width, 5 μm) from the general mean for both of these types of vessels. Results indicate that the primary site of oxygen delivery to tissue is located between the smallest arterioles and venules (change of 16.3 mmHg, P = 0.001). In conclusion, oxygen losses from mesenteric arterioles and venules are negligible, indicating low metabolic rates for both the vascular wall and the mesenteric tissue. Capillaries appear to be the primary site of oxygen delivery to the tissue in the mesenteric microcirculation. In light of the present results, previously reported data concerning oxygen consumption in the mesenteric microcirculation can be explained as artifacts of accumulated oxygen consumption due to the application of instrumentation having a large excitation area for Po2measurements in slow moving and stationary media.
Sympathetic control of heart rate in nNOS knockout mice
Authors: Choate J. K. , Murphy S. M. , Feldman R. , Anderson C. R.
Published: October 2007

Inhibition of neuronal nitric oxide synthase (nNOS) in cardiac postganglionic sympathetic neurons leads to enhanced cardiac sympathetic responsiveness in normal animals, as well as in animal models of cardiovascular diseases. We used isolated atria from mice with selective genetic disruption of nNOS (nNOS−/−) and their wild-type littermates (WT) to investigate whether sympathetic heart rate (HR) responses were dependent on nNOS. Immunohistochemistry was initially used to determine the presence of nNOS in sympathetic [tyrosine hydroxylase (TH) immunoreactive] nerve terminals in the mouse sinoatrial node (SAN). After this, the effects of postganglionic sympathetic nerve stimulation (1–10 Hz) and bath-applied norepinephrine (NE; 10−8–10−4 mol/l) on HR were examined in atria from nNOS−/− and WT mice. In the SAN region of WT mice, TH and nNOS immunoreactivity was virtually never colocalized in nerve fibers. nNOS−/− atria showed significantly reduced HR responses to sympathetic nerve activation and NE ( P < 0.05). Similarly, the positive chronotropic response to the adenylate cyclase activator forskolin (10−7–10−5 mol/l) was attenuated in nNOS−/− atria ( P < 0.05). Constitutive NOS inhibition with l-nitroarginine (0.1 mmol/l) did not affect the sympathetic HR responses in nNOS−/− and WT atria. The paucity of nNOS in the sympathetic innervation of the mouse SAN, in addition to the attenuated HR responses to neuronal and applied NE, indicates that presynaptic sympathetic neuronal NO does not modulate neuronal NE release and SAN pacemaking in this species. It appears that genetic deletion of nNOS results in the inhibition of adrenergic-adenylate cyclase signaling within SAN myocytes.
Increased mitochondrial calcium coexists with decreased reperfusion injury in postconditioned (but not preconditioned) hearts
Authors: Argaud Laurent , Gateau-Roesch Odile , Augeul Lionel , Couture-Lepetit Elisabeth , Loufouat Joseph , Gomez Ludovic , Robert Dominique , Ovize Michel
Published: October 2007

Ca2+ is the main trigger for mitochondrial permeability transition pore opening, which plays a key role in cardiomyocyte death after ischemia-reperfusion. We investigated whether a reduced accumulation of mitochondrial Ca2+ might explain the attenuation of lethal reperfusion injury by postconditioning. Anesthetized New Zealand White rabbits underwent 30 min of ischemia, followed by either 240 (infarct size protocol) or 60 (mitochondria protocol) min of reperfusion. They received either no intervention (control), preconditioning by 5-min ischemia and 5-min reperfusion, postconditioning by four cycles of 1-min reperfusion and 1-min ischemia at the onset of reflow, or pharmacological inhibition of the transition pore opening by N-methyl-4-isoleucine-cyclosporin (NIM811; 5 mg/kg iv) given at reperfusion. Area at risk and infarct size were assessed by blue dye injection and triphenyltetrazolium chloride staining. Mitochondria were isolated from the risk region for measurement of 1) Ca2+ retention capacity (CRC), and 2) mitochondrial content of total (atomic absorption spectrometry) and ionized (potentiometric technique) calcium concentration. CRC averaged 0.73 ± 0.16 in control vs. 4.23 ± 0.17 μg Ca2+/mg proteins in shams ( P < 0.05). Postconditioning, preconditioning, or NIM811 significantly increased CRC ( P < 0.05 vs. control). In the control group, total and free mitochondrial calcium significantly increased to 2.39 ± 0.43 and 0.61 ± 0.10, respectively, vs. 1.42 ± 0.09 and 0.16 ± 0.01 μg Ca2+/mg in sham ( P < 0.05). Surprisingly, whereas total and ionized mitochondrial Ca2+ decreased in preconditioning, it significantly increased in postconditioning and NIM811 groups. These data suggest that retention of calcium within mitochondria may explain the decreased reperfusion injury in postconditioned (but not preconditioned) hearts.
The role of the receptor for advanced glycation end-products in lung fibrosis
Authors: He Mei , Kubo Hiroshi , Ishizawa Kota , Hegab Ahmed E. , Yamamoto Yasuhiko , Yamamoto Hiroshi , Yamaya Mutsuo
Published: October 2007

The pathogenesis of pulmonary fibrosis remains unclear. The receptor for advanced glycation end-products (RAGE) is a multi-ligand receptor known to be involved in the process of fibrotic change in several organs, such as peritoneal fibrosis and kidney fibrosis. The aim of this study was to examine the contribution of RAGE during the acute inflammation and chronic fibrotic phases of lung injury induced by intratracheal instillation of bleomycin in mice. Bleomycin-induced lung fibrosis was evaluated in wild-type and RAGE-deficient (RAGE−/−) mice. Bleomycin administration to wild-type mice caused an initial pneumonitis that evolved into fibrosis. While RAGE−/− mice developed a similar early inflammatory response, the mice were largely protected from the late fibrotic effects of bleomycin. The protection afforded by RAGE deficiency was accompanied by reduced pulmonary levels of the potent RAGE-inducible profibrotic cytokines transforming growth factor (TGF)-β and PDGF. In addition, bleomycin administration induced high mobility group box 1 (HMGB-1) production, one of the ligands of RAGE, from inflammatory cells that accumulated within the air space. Coculture with HMGB-1 induced epithelial-mesenchymal transition (EMT) in alveolar type II epithelial cells from wild-type mice. However, alveolar type II epithelial cells derived from RAGE−/− mice did not respond to HMGB-1 treatment, such that the RAGE/HMGB-1 axis may play an important role in EMT. Also, bleomycin administration induced profibrotic cytokines TGF-β and PDGF only in wild-type mouse lungs. Our results suggested that RAGE contributes to bleomycin-induced lung fibrosis through EMT and profibrotic cytokine production. Thus, RAGE may be a new therapeutic target for pulmonary fibrosis.
Role of platelet-activating factor in pulmonary vascular remodeling associated with chronic high altitude hypoxia in ovine fetal lambs
Authors: Bixby Christine E. , Ibe Basil O. , Abdallah May F. , Zhou Weilin , Hislop Alison A. , Longo Lawrence D. , Raj J. Usha
Published: October 2007

Platelet-activating factor (PAF) is implicated in pathogenesis of chronic hypoxia-induced pulmonary hypertension in some animal models and in neonates. Effects of chronic hypoxia on PAF receptor (PAF-R) system in fetal pulmonary vasculature are unknown. We investigated the effect of chronic high altitude hypoxia (HAH) in fetal lambs [pregnant ewes were kept at 3,801 m (12,470 ft) altitude from ∼35 to 145 days gestation] on PAF-R-mediated effects in the pulmonary vasculature. Age-matched controls were kept at sea level. Intrapulmonary arteries were isolated, and smooth muscle cells (SMC-PA) were cultured from HAH and control fetuses. To determine presence of pulmonary vascular remodeling, lung tissue sections were subjected to morphometric analysis. Percentage medial wall thickness was significantly increased ( P < 0.05) in arteries at all levels in the HAH lambs. PAF-R protein expression studied by immunocytochemistry and Western blot analysis on lung tissue SMC-PA demonstrated greater PAF-R expression in HAH lambs. PAF-R binding (femtomoles per 106cells) in HAH SMC-PA was 90.3 ± 4.08 and 66% greater than 54.3 ± 4.9 in control SMC-PA. Pulmonary arteries from HAH fetuses synthesized >3-fold PAF than vessels from controls. Compared with controls SMC-PA of HAH lambs demonstrated 139% and 40% greater proliferation in 10% FBS alone and with 10 nM PAF, respectively. Our data demonstrate that exposure of ovine fetuses to HAH will result in significant upregulation of PAF synthesis, PAF-R expression, and PAF-R-mediated effects in pulmonary arteries. These findings suggest that increased PAF-R protein expression and increased PAF binding contribute to pulmonary vascular remodeling in these animals and may predispose them to persistent pulmonary hypertension after birth.
Spleen and cardiovascular function during short apneas in divers
Authors: Palada Ivan , Eterović Davor , Obad Ante , Bakovic Darija , Valic Zoran , Ivancev Vladimir , Lojpur Mihajlo , Shoemaker J. Kevin , Dujic Zeljko
Published: October 2007

We investigated the spleen volume changes as related to the cardiovascular responses during short-duration apneas at rest. We used dynamic ultrasound splenic imaging and noninvasive photoplethysmographic cardiovascular measurements before, during, and after 15–20 s apneas in seven trained divers. The role of baroreflex was studied by intravenous bolus of vasodilating drug trinitrosan during tidal breathing. The role of lung volume was studied by comparing the apneas at near-maximal lung volume with apneas after inhaling tidal volume, with and without cold forehead stimulation. In apneas at near maximal lung volume, a 20% reduction in splenic volume ( P = 0.03) was observed as early as 3 s after the onset of breath holding. Around that time the heart rate increased, the mean arterial pressure abruptly decreased from 89.6 to 66.7 mmHg ( P = 0.02), and cardiac output decreased, on account of reduction in stroke volume. Intravenous application of trinitrosan resulted in ∼6-mmHg decrement in mean arterial pressure, while the splenic volume decreased for ∼13%. In apneas at low lung volume, the early splenic contraction was also observed, 10% without and 12% with cold forehead stimulation, although the mean arterial pressure did not change or even increased, respectively. In conclusion, the spleen contraction is present at the beginning of apnea, accentuated by cold forehead stimulation. At large, but not small, lung volume, this initial contraction is probably facilitated by downloaded baroreflex in conditions of decreased blood pressure and cardiac output.
The acute effects of differential dietary fatty acids on human skeletal muscle pyruvate dehydrogenase activity
Authors: Bradley Nicolette S. , Heigenhauser George J. F. , Roy Brian D. , Staples Elizabeth M. , Inglis J. Greig , LeBlanc Paul J. , Peters Sandra J.
Published: October 2007

Pyruvate dehydrogenase (PDH) is an important regulator of carbohydrate oxidation during exercise, and its activity can be downregulated by an increase in dietary fat. The purpose of this study was to determine the acute metabolic effects of differential dietary fatty acids on the activation of the PDH complex (PDHa activity) at rest and at the onset of moderate-intensity exercise. University-aged male subjects ( n = 7) underwent two fat-loading trials spaced at least 2 wk apart. Subjects consumed ∼300 g saturated (SFA) or n-6 polyunsaturated fatty acid (PUFA) fat over the course of 5 h. Following this, participants cycled at 65% of their maximum oxygen uptake for 15 min. Muscle biopsies were taken before and following fat loading and at 1 min exercise. Plasma free fatty acids increased from 0.15 ± 0.07 to 0.54 ± 0.19 mM over 5 h with SFA and from 0.11 ± 0.04 to 0.35 ± 0.13 mM with n-6 PUFA and were significantly lower throughout the n-6 PUFA trial. PDHa activity was unchanged following fat loading but increased at the onset of exercise in the SFA trial, from 1.18 ± 0.27 to 2.16 ± 0.37 mmol·min−1·kg wet wt−1. This effect was negated in the n-6 PUFA trial (1.04 ± 0.20 to 1.28 ± 0.36 mmol·min−1·kg wet wt−1). PDH kinase was unchanged in both trials, suggesting that the attenuation of PDHa activity with n-6 PUFA was a result of changes in the concentrations of intramitochondrial effectors, potentially intramitochondrial NADH or Ca2+. Our findings suggest that attenuated PDHa activity contributes to the preferential oxidation of n-6 PUFA during moderate-intensity exercise.
Differential effects of chronic hypoxia and intermittent hypocapnic and eucapnic hypoxia on pulmonary vasoreactivity
Authors: Snow Jessica B. , Kitzis Vanessa , Norton Charles E. , Torres Samantha N. , Johnson Kimberly D. , Kanagy Nancy L. , Walker Benjimen R. , Resta Thomas C.
Published: October 2007

Intermittent hypoxia (IH) resulting from sleep apnea can lead to pulmonary hypertension (PH) and right heart failure, similar to chronic sustained hypoxia (CH). Supplemental CO2, however, attenuates hypoxic PH. We therefore hypothesized that, similar to CH, IH elicits PH and associated increases in arterial endothelial nitric oxide synthase (eNOS) expression, ionomycin-dependent vasodilation, and receptor-mediated pulmonary vasoconstriction. We further hypothesized that supplemental CO2inhibits these responses to IH. To test these hypotheses, we measured eNOS expression by Western blot in intrapulmonary arteries from CH (2 wk, 0.5 atm), hypocapnic IH (H-IH) (3 min cycles of 5% O2/air flush, 7 h/day, 2 wk), and eucapnic IH (E-IH) (3 min cycles of 5% O2, 5% CO2/air flush, 7 h/day, 2 wk) rats and their respective controls. Furthermore, vasodilatory responses to the calcium ionophore ionomycin and vasoconstrictor responses to the thromboxane mimetic U-46619 were measured in isolated saline-perfused lungs from each group. Hematocrit, arterial wall thickness, and right ventricle-to-total ventricle weight ratios were additionally assessed as indexes of polycythemia, arterial remodeling, and PH, respectively. Consistent with our hypotheses, E-IH resulted in attenuated polycythemia, arterial remodeling, RV hypertrophy, and eNOS upregulation compared with H-IH. However, in contrast to CH, neither H-IH nor E-IH increased ionomycin-dependent vasodilation. Furthermore, H-IH and E-IH similarly augmented U-46619-induced pulmonary vasoconstriction but to a lesser degree than CH. We conclude that maintenance of eucapnia decreases IH-induced PH and upregulation of arterial eNOS. In contrast, increases in pulmonary vasoconstrictor reactivity following H-IH are unaltered by exposure to supplemental CO2.
Compliance, actuation, and work characteristics of the goat foreleg and hindleg during level, uphill, and downhill running
Authors: Lee David V. , McGuigan M. Polly , Yoo Edwin H. , Biewener Andrew A.
Published: October 2007

We model the action of muscle-tendon system(s) about a given joint as a serial actuator and spring. By this technique, the experimental joint moment is imposed while the combined angular deflection of the actuator and spring are constrained to match the experimental joint angle throughout the stance duration. The same technique is applied to the radial leg (i.e., shoulder/hip-to-foot). The spring constant that minimizes total actuator work is considered optimal, and this minimum work is expressed as a fraction of total joint/radial leg work, yielding an actuation ratio (AR; 1 = pure actuation and 0 = pure compliance). To address work modulation, we determined the specific net work (SNW), the absolute value of net divided by total work. This ratio is unity when only positive or negative work is done and zero when equal energy is absorbed and returned. Our proximodistal predictions of joint function are supported during level and 15° grade running. The greatest AR and SNW are found in the proximal leg joints (elbow and knee). The ankle joint is the principal spring of the hindleg and shows no significant change in SNW with grade, reflecting the true compliance of the common calcaneal tendon. The principal foreleg spring is the metacarpophalangeal joint. The observed pattern of proximal actuation and distal compliance, as well as the substantial SNW at proximal joints, minimal SNW at intermediate joints, and variable energy absorption at distal joints, may emerge as general principles in quadruped limb mechanics and help to inform the leg designs of highly capable running robots.
Mouse Embryo Fibroblasts Lacking the Tumor Suppressor Menin Show Altered Expression of Extracellular Matrix Protein Genes
Authors: Ji Youngmi , Prasad Nijaguna B. , Novotny Elizabeth A. , Kaur Sukhbir , Elkahloun Abdel , Chen Yidong , Zhang Rui-Zhu , Chu Mon-Li , Agarwal Sunita K. , Marx Stephen J. , Collins Francis S. , Chandrasekharappa Settara C.
Published: October 2007

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant familial cancer syndrome characterized primarily by endocrine tumors of the parathyroids, anterior pituitary, and enteropancreatic endocrine tissues. Affected individuals carry a germ-line loss-of-function mutation of the MEN1 gene, and tumors arise after loss of the second allele. Homozygous loss of Men1 in the germ line of mice results in early embryonic lethality, with defective development of neural tube, heart, liver, and craniofacial structures. We generated immortalized wild-type (WT) and menin-null mouse embryo fibroblast (MEF) cell lines and evaluated their characteristics, including global expression patterns. The WT and menin-null cell lines were aneuploid, and the nulls did not display tumorigenic characteristics in soft agar assay. Expression arrays in menin-null MEFs revealed altered expression of several extracellular matrix proteins that are critical in organogenesis. Specifically, transcripts for fibulin 2 (Fbln2), periostin (Postn), and versican [chondroitin sulfate proteoglycan (Cspg2)], genes critical for the developing heart and known to be induced by transforming growth factor-β (TGF-β), were decreased in their expression in menin-null MEFs. Fbln2 expression was the most affected, and the reduction in menin-null MEFs for Fbln2, Postn, and Cspg2 was 16.18-, 5.37-, and 2.15-fold, respectively. Menin-null MEFs also showed poor response to TGF-β–induced Smad3-mediated transcription in a reporter assay, supporting a role for menin in this pathway. Postn and Cspg2 expression in WT, unlike in null MEFs, increased on TGF-β treatment. The expression changes associated with the loss of the tumor suppressor menin provide insights into the defective organogenesis observed during early embryonic development in Men1-null mouse embryos. (Mol Cancer Res 2007;5(10):1041–51)
Abelson Interactor Protein-1 Positively Regulates Breast Cancer Cell Proliferation, Migration, and Invasion
Authors: Wang Chunjie , Navab Roya , Iakovlev Vladimir , Leng Yan , Zhang Jinyi , Tsao Ming-Sound , Siminovitch Katherine , McCready David R. , Done Susan J.
Published: October 2007

Abelson interactor protein-1 (ABI-1) is an adaptor protein involved in actin reorganization and lamellipodia formation. It forms a macromolecular complex containing Hspc300/WASP family verprolin-homologous proteins 2/ABI-1/nucleosome assembly protein 1/PIR121 or Abl/ABI-1/WASP family verprolin-homologous proteins 2 in response to Rho family-dependent stimuli. Due to its role in cell mobility, we hypothesized that ABI-1 has a role in invasion and metastasis. In the present study, we found that weakly invasive breast cancer cell lines (MCF-7, T47D, MDA-MB-468, SKBR3, and CAMA1) express lower levels of ABI-1 compared with highly invasive breast cancer cell lines (MDA-MB-231, MDA-MB-157, BT549, and Hs578T), which exhibit high ABI-1 levels. Using RNA interference, ABI-1 was stably down-regulated in MDA-MB-231, which resulted in decreased cell proliferation and anchorage-dependent colony formation and abrogation of lamellipodia formation on fibronectin. Down-regulation of ABI-1 decreased invasiveness and migration ability and decreased adhesion on collagen IV and actin polymerization in MDA-MB-231 cells. Additionally, compared with control parental cells, ABI-1 small interfering RNA–transfected cells showed decreased levels of phospho-PDK1, phospho-Raf, phospho-AKT, total AKT, and AKT1. These data suggest that ABI-1 plays an important role in the spread of breast cancer and that this role may be mediated via the phosphatidylinositol 3-kinase pathway. (Mol Cancer Res 2007;5(10):1031–9)
Human 8-Oxoguanine DNA Glycosylase Suppresses the Oxidative Stress–Induced Apoptosis through a p53-Mediated Signaling Pathway in Human Fibroblasts
Authors: Youn Cha-Kyung , Song Peter I. , Kim Mi-Hwa , Kim Jin Sook , Hyun Jin-Won , Choi Sang-Joon , Yoon Sang Pil , Chung Myung Hee , Chang In-Youb , You Ho Jin
Published: October 2007

Human 8-oxoguanine DNA glycosylase (hOGG1) is the main defense enzyme against mutagenic effects of cellular 7,8-dihydro-8-oxoguanine. In this study, we investigated the biological role of hOGG1 in DNA damage–related apoptosis induced by hydrogen peroxide (H2O2)–derived oxidative stress. The down-regulated expression of hOGG1 by its small interfering RNA prominently triggers the H2O2-induced apoptosis in human fibroblasts GM00637 and human lung carcinoma H1299 cells via the p53-mediated apoptotic pathway. However, the apoptotic responses were specifically inhibited by hOGG1 overexpression. The p53–small interfering RNA transfection into the hOGG1-deficient GM00637 markedly inhibited the H2O2-induced activation of p53-downstream target proteins such as p21, Noxa, and caspase-3/7, which eventually resulted in the increased cell viability. Although the cell viability of hOGG1-knockdown H1299 p53 null cells was similar to that of the hOGG1 wild-type H1299, after the overexpression of p53 the hOGG1-knockdown H1299 showed the significantly decreased cell viability compared with that of the hOGG1 wild-type H1299 at the same experimental condition. Moreover, the array comparative genome hybridization analyses revealed that the hOGG1-deficient GM00637 showed more significant changes in the copy number of large regions of their chromosomes in response to H2O2 treatment. Therefore, we suggest that although p53 is a major modulator of apoptosis, hOGG1 also plays a pivotal role in protecting cells against the H2O2-induced apoptosis at the upstream of the p53-dependent pathway to confer a survival advantage to human fibroblasts and human lung carcinomas through maintaining their genomic stability. (Mol Cancer Res 2007;5(10):1083–98)
Fas-Negative Osteosarcoma Tumor Cells Are Selected during Metastasis to the Lungs: The Role of the Fas Pathway in the Metastatic Process of Osteosarcoma
Authors: Koshkina Nadezhda V. , Khanna Chand , Mendoza Arnulfo , Guan Hui , DeLauter Lindsey , Kleinerman Eugenie S.
Published: October 2007

Low expression of Fas by different tumors including osteosarcoma, correlates with poor prognosis. We found that osteosarcoma lung metastases from patients expressed negligible amounts of Fas, but primary tumors often expressed high Fas levels. The reason for this discrepancy is unknown. We hypothesized that because FasL is constitutively expressed in the lungs, Fas-positive (Fas+) tumor cells entering the lungs would bind with FasL and die from Fas-induced apoptosis, resulting in the “selection” of Fas-negative (Fas−) cells, which would eventually form metastases. To test this hypothesis, we injected K7 osteosarcoma cells, which express functional Fas in vitro, into mice and confirmed that its bone tumors were Fas+, but lung metastases were Fas−. Next, to inhibit Fas signaling without affecting Fas expression, we transfected these cells with a FADD-dominant negative (FDN) plasmid and developed K7/FDN cells. Metastases formed by K7/FDN cells contained Fas+ tumor cells. Moreover, K7/FDN cells were retained in the lungs longer and formed more lung metastases than K7 cells. In addition, the incidence of lung metastases in FasL-deficient mice injected with K7 cells was higher than that in wild-type mice. Metastases from FasL-deficient mice but not from wild-type mice contained Fas+ tumor cells. Based on that, we conclude that Fas− osteosarcoma cells are selected during lung metastases formation and that inhibition of Fas signaling in tumors or lack of FasL in the host environment allows the proliferation of Fas+ osteosarcoma cells in the lungs and promotes metastases growth. Therefore, Fas may be considered as a new therapeutic target for osteosarcoma treatment. (Mol Cancer Res 2007;5(10):991–9)
Oligodendrocyte Lineage Transcription Factor 2 Inhibits the Motility of a Human Glial Tumor Cell Line by Activating RhoA
Authors: Tabu Kouichi , Ohba Yusuke , Suzuki Tadaki , Makino Yoshinori , Kimura Taichi , Ohnishi Akiko , Sakai Mieko , Watanabe Takuya , Tanaka Shinya , Sawa Hirofumi
Published: October 2007

The basic helix-loop-helix transcription factor, oligodendrocyte lineage transcription factor 2 (OLIG2), is specifically expressed in the developing and mature central nervous system and plays an important role in oligodendrogenesis from neural progenitors. It is also expressed in various types of glial tumors, but rarely in glioblastoma. Although we previously showed that OLIG2 expression inhibits glioma cell growth, its role in tumorigenesis remains incompletely understood. Here, we investigated the effect of OLIG2 expression on the migration of the human glioblastoma cell line U12-1. In these cells, OLIG2 expression is controlled by the Tet-off system. Induction of OLIG2 expression inhibited both the migration and invasiveness of U12-1 cells. OLIG2 expression also increased the activity of the GTPase RhoA as well as inducing the cells to form stress fibers and focal adhesions. Experiments using short interfering RNA against p27Kip1 revealed that up-regulation of the p27Kip1 protein was not essential for RhoA activation, rather it contributed independently to the decreased motility of OLIG2-expressing U12-1 cells. Alternatively, semiquantitative reverse transcription-PCR analysis revealed that mRNA expression of RhoGAP8, which regulates cell migration, was decreased by OLIG2 expression. Furthermore, expression of C3 transferase, which inhibits Rho via ADP ribosylation, attenuated the OLIG2-induced inhibition of cell motility. Imaging by fluorescence resonance energy transfer revealed that in U12-1 cells lacking OLIG2, the active form of RhoA was localized to protrusions of the cell membrane. In contrast, in OLIG2-expressing cells, it lined almost the entire plasma membrane. Thus, OLIG2 suppresses the motile phenotype of glioblastoma cells by activating RhoA. (Mol Cancer Res 2007;5(10):1099–109)
Hepatitis B Virus Pre-S2 Mutant Surface Antigen Induces Degradation of Cyclin-Dependent Kinase Inhibitor p27Kip1 through c-Jun Activation Domain-Binding Protein 1
Authors: Hsieh Yi-Hsuan , Su Ih-Jen , Wang Hui-Ching , Tsai Jui-He , Huang Yu-Jun , Chang Wen-Wei , Lai Ming-Derg , Lei Huan-Yaw , Huang Wenya
Published: October 2007

The hepatitis B virus (HBV) large surface antigen (LHBS) mutant with deletion at the pre-S2 region accumulates in endoplasmic reticulum (ER) and is associated with HBV-induced hepatocellular carcinogenesis. In this study, we found that the pre-S2 LHBS mutant directly interacts with the Jun activation domain–binding protein 1 (JAB1). Association of pre-S2 LHBS with JAB1 dissociated JAB1 from the JAB1/IRE1 complex in ER. The free (active) JAB1 then translocated into cell nuclei and rendered the Cdk inhibitor p27Kip1 to cytosolic proteasome for degradation. The pre-S2 LHBS mutant induced hyperphosphorylation of tumor suppressor retinoblastoma (RB) via cyclin-dependent kinase 2 (Cdk2), a downstream molecule regulated by p27Kip1. This effect is independent of the ER stress signaling pathway. The transgenic mice carrying the pre-S2 mutant LHBS gene also exhibited Cdk2 activation, p27Kip1 degradation, as well as RB hyperphosphorylation. The mouse hepatocytes exhibited morphologic abnormalities such as chromatin condensation, multinucleation, and dysplasia of hepatocytes. In summary, the pre-S2 LHBS mutant causes p27Kip1 degradation through direct interaction with JAB1. The pre-S2 mutant LHBS is suggested to be a potential oncoprotein for HBV-related hepatocellular carcinoma. (Mol Cancer Res 2007;5(10):1063–72)