bluuebunny/crossref_metadata_2025 huggingface dataset:
Four‐dimensional cone‐beam computed tomography using an on‐board imager
Authors: Li Tianfang , Xing Lei , Munro Peter , McGuinness Christopher , Chao Ming , Yang Yong , Loo Bill , Koong Albert
Published: October 2006

On‐board cone‐beam computed tomography (CBCT) has recently become available to provide volumetric information of a patient in the treatment position, and holds promises for improved target localization and irradiation dose verification. The design of currently available on‐board CBCT, however, is far from optimal. Its quality is adversely influenced by many factors, such as scatter, beam hardening, and intra‐scanning organ motion. In this work we quantitatively study the influence of organ motion on CBCT imaging and investigate a strategy to acquire high quality phase‐resolved [four‐dimensional (4D)] CBCT images based on phase binning of the CBCT projection data. An efficient and robust method for binning CBCT data according to the patient's respiratory phase derived in the projection space was developed. The phase‐binned projections were reconstructed using the conventional Feldkamp algorithm to yield 4D CBCT images. Both phantom and patient studies were carried out to validate the technique and to optimize the 4D CBCT data acquisition protocol. Several factors that are important to the clinical implementation of the technique, such as the image quality, scanning time, number of projections, and radiation dose, were analyzed for various scanning schemes. The general references drawn from this study are: (i) reliable phase binning of CBCT projections is accomplishable with the aid of external or internal marker and simple analysis of its trace in the projection space, and (ii) artifact‐free 4D CBCT images can be obtained without increasing the patient radiation dose as compared to the current 3D CBCT scan.
Classifying undetermined poisoning deaths
Authors: Donaldson A E , Larsen G Y , Fullerton-Gleason L , Olson L M
Published: October 2006

Objective:To classify poisoning deaths of undetermined intent as either suicide or unintentional and to estimate the extent of underreported poisoning suicides.

Methods:Based on 2002 statewide death certificate and medical examiner data in Utah, the authors randomly selected one half of undetermined and unintentional poisoning deaths for data abstraction and included all suicides. Bivariate analyses assessed differences in demographics, death characteristics, forensic toxicology results, mental health history, and other potentially contributing factors. Classification and regression tree (CART) analysis used information from unintentional and suicide poisoning deaths to create a classification tree that was applied to undetermined poisoning deaths.

Results:The authors analyzed 41 unintentional, 87 suicide, and 84 undetermined poisonings. Undetermined and unintentional decedents were similar in the presence of opiates, physical health problems, and drug abuse. Although none of the undetermined decedents left a suicide note, previous attempt or intent to commit suicide was reported for 11 (13%) of these cases. CART analysis identified suicidal behavior, drug abuse, physical health problems, depressed mood, and age as discriminating between suicide and unintentional poisoning. It is estimated that suicide rates related to poisoning are underreported by approximately 30% and overall suicide rates by 10%. Unintentional poisoning death rates were underreported by 61%.

Conclusions:This study suggests that manner of death determination relies on circumstance dependent variables that may not be consistently captured by medical examiners. Underreporting of suicide rates has important implications in policy development, research funding, and evaluation of prevention programs.
The influence of local politicians on pedestrian safety
Authors: Lyons R A , Jones S J , Newcombe R G , Palmer S R
Published: October 2006

Aim:To determine whether local politicians influence the distribution of traffic calming measures.

Methods:Longitudinal ecological study in two UK cities. Local political constituencies were categorized by representation by members of the cabinet structure as a marker of influence. The density of traffic calming features per political area, adjusted for the historical pattern of road injuries, was compared between cabinet represented and non-represented areas.

Results:Traffic calming density was significantly associated with cabinet representation status, adjusted for historical collision risk (risk ratio 2.77, 95% confidence interval 1.37 to 5.61).

Conclusion:These results support the hypothesis that senior local politicians are effective advocates for enhancing safety in their areas.
Acute modulation of PP2a and troponin I phosphorylation in ventricular myocytes: studies with a novel PP2a peptide inhibitor
Authors: Deshmukh Prajwal A. , Blunt Bradford C. , Hofmann Polly A.
Published: September 2006

The present study demonstrates that acute activation with either β-adrenergic receptor agonists or H2O2treatment increases protein phosphatase 2a (PP2a) activity in ventricular myocytes. PP2a activation occurs concomitant with an increase in methylation of PP2a, changes in localization of a PP2a targeting subunit PP2aB56α, and a decrease in phosphorylation of PP2a substrates, such as troponin I (TnI) and ERK in ventricular myocytes. Okadaic acid, a well-established pharmacological inhibitor of PP2a, and the peptide Thr-Pro-Asp-Tyr-Phe-Leu (TPDYFL) were used to block PP2a methylation, localization, and phosphorylations. TPDYFL is a highly conserved sequence of the PP2a catalytic subunit COOH-terminus. Specifically, both okadaic acid and the peptide increased β-adrenergic-cAMP-dependent phosphorylation of TnI and blocked the β-adrenergic-cAMP-dependent translocation of PP2aB56α. TPDYFL, but not a scrambled version of this sequence, blocked H2O2-induced changes in PP2a methylation and TnI dephosphorylation. Okadaic acid produces similar inhibition of H2O2effects. Thus we propose that the novel peptide TPDYFL acts as an inhibitor of PP2a activity and may be a useful tool to increase our understanding of how PP2a is regulated and the role of PP2a in a variety of physiological and pathological processes. In addition, the present study is consistent with acute β-adrenergic receptor activation and H2O2exposure, simultaneously activating kinases and PP2a to work on common substrates, such as TnI. We hypothesize that dual activation of opposing enzymes provides for a tighter regulation of substrate phosphorylations in ventricular myocytes.
Interstitial K+ concentration in active muscle after myocardial infarction
Authors: Li Jianhua , Gao Zhaohui , Kehoe Valerie , Sinoway Lawrence I.
Published: September 2006

Previous work demonstrated that Na+-K+ pump activity within skeletal muscle is attenuated in myocardial infarction (MI). This may lead to enhanced interstitial K+ concentration ([K+]o) in the muscle. We tested the hypothesis that [K+]o rises with muscle contraction and that, in rats with MI, the rate of rise in [K+]o is greater than it is in control animals. Microdialysis probes were inserted in the skeletal muscle of six healthy control and six MI rats. The ends of the probes were then attached to the K+ electrodes, and [K+]o was continuously measured. Muscle contraction was induced by electrical stimulation of the sciatic nerves for 1 min. Stimulation at 1 and 3 Hz increased muscle [K+]o by 14.2% and 44.7% in controls and by 22.9% and 62.8% in MI rats ( P < 0.05 vs. controls), respectively. When ouabain, an inhibitor of Na+-K+ pump, was added to the perfusate, muscle [K+]o rose significantly. This effect of ouabain was significantly attenuated in MI animals. In conclusion, when compared with that in control animals, an increase of [K+]o in exercising muscle is augmented in MI rats, likely due to an attenuation of Na+-K+ pump activity.
Intracellular sodium determines frequency-dependent alterations in contractility in hypertrophied feline ventricular myocytes
Authors: Mills Geoffrey D. , Harris David M. , Chen Xiongwen , Houser Steven R.
Published: September 2006

Hypertrophy and failure (H/F) in humans and large mammals are characterized by a change from a positive developed force-frequency relationship (+FFR) in normal myocardium to a flattened or negative developed force-frequency relationship (−FFR) in disease. Altered Ca2+ homeostasis underlies this process, but the role of intracellular Na+ concentration ([Na+]i) in H/F and frequency-dependent contractility reserve is unclear. We hypothesized that altered [Na+]i is central to the −FFR response in H/F feline myocytes. Aortic constriction caused left ventricular hypertrophy (LVH). We found that as pacing rate was increased, contraction magnitude was maintained in isolated control myocytes (CM) but decreased in LVH myocytes (LVH-M). Quiescent LVH-M had higher [Na+]i than CM (LVH-M 13.3 ± 0.3 vs. CM 8.9 ± 0.2 mmol/l; P < 0.001) with 0.5-Hz pacing (LVH-M 14.9 ± 0.5 vs. CM 10.8 ± 0.4 mmol/l; P < 0.001) but were not different at 2.5 Hz (17.0 ± 0.7 vs. control 16.0 ± 0.7 mmol/l; not significant). [Na+]i was altered by patch pipette dialysis to define the effect of [Na+]i on contraction magnitude and action potential (AP) wave shape at slow and fast pacing rates. Using AP clamp, we showed that LVH-M require increased [Na+]i and long diastolic intervals to maintain normal shortening. Finally, we determined the voltage dependence of contraction for Ca2+ current ( ICa)-triggered and Na+/Ca2+ exchanger-mediated contractions and showed that there is a greater [Na+]i dependence of contractility in LVH-M. These data show that increased [Na+]i is essential for maintaining contractility at slow heart rates but contributes to small contractions at fast rates unless rate-dependent AP shortening is prevented, suggesting that altered [Na+]i regulation is a critical contributor to abnormal contractility in disease.
Nitric oxide preconditioning regulates endothelial monolayer integrity via the heat shock protein 90-soluble guanylate cyclase pathway
Authors: Antonova Galina N. , Snead Connie M. , Antonov Alexander S. , Dimitropoulou Christiana , Venema Richard C. , Catravas John D.
Published: September 2006

Large (pathological) amounts of nitric oxide (NO) induce cell injury, whereas low (physiological) NO concentrations often ameliorate cell injury. We tested the hypotheses that pretreatment of endothelial cells with low concentrations of NO (preconditioning) would prevent injury induced by high NO concentrations. Apoptosis, induced in bovine aortic endothelial cells (BAECs) by exposing them to either 4 mM sodium nitroprusside (SNP) or 0.5 mM N-(2-aminoethyl)- N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate) for 8 h, was abolished by 24-h pretreatment with either 100 μM SNP, 10 μM spermine NONOate, or 100 μM 8-bromo-cGMP (8-Br-cGMP). Repair of BAECs following wounding, measured as the recovery rate of transendothelial electrical resistance, was delayed by 8-h exposure to 4 mM SNP, and this delay was significantly attenuated by 24-h pretreatment with 100 μM SNP. NO preconditioning produced increased association and expression of soluble guanyl cyclase (sGC) and heat shock protein 90 (HSP90). The protective effect of NO preconditioning, but not the injurious effect of 4 mM SNP, was abolished by either a sGC activity inhibitor 1H-[1,2,4]oxadiazolo-[4,3- a]quinoxalin-1-one (ODQ) or a HSP90 binding inhibitor (radicicol) and was mimicked by 8-Br-cGMP. We conclude that preconditioning with a low dose of NO donor accelerates repair and maintains endothelial integrity via a mechanism that includes the HSP90/sGC pathway. HSP90/sGC may thus play a role in the protective effects of NO-generating drugs from injurious stimuli.
Blocking cardiac growth in hypertrophic cardiomyopathy induces cardiac dysfunction and decreased survival only in males
Authors: Luckey Stephen W. , Mansoori Jason , Fair Kelly , Antos Christopher L. , Olson Eric N. , Leinwand Leslie A.
Published: September 2006

Mutations in myosin heavy chain (MyHC) can cause hypertrophic cardiomyopathy (HCM) that is characterized by hypertrophy, histopathology, contractile dysfunction, and sudden death. The signaling pathways involved in the pathology of HCM have not been elucidated, and an unresolved question is whether blocking hypertrophic growth in HCM may be maladaptive or beneficial. To address these questions, a mouse model of HCM was crossed with an antihypertrophic mouse model of constitutive activated glycogen synthase kinase-3β (caGSK-3β). Active GSK-3β blocked cardiac hypertrophy in both male and female HCM mice. However, doubly transgenic males (HCM/GSK-3β) demonstrated depressed contractile function, reduced sarcoplasmic (endo) reticulum Ca2+-ATPase (SERCA) expression, elevated atrial natriuretic factor (ANF) expression, and premature death. In contrast, female HCM/GSK-3β double transgenic mice exhibited similar cardiac histology, function, and survival to their female HCM littermates. Remarkably, dietary modification from a soy-based diet to a casein-based diet significantly improved survival in HCM/GSK-3β males. These findings indicate that activation of GSK-3β is sufficient to limit cardiac growth in this HCM model and the consequence of caGSK-3β was sexually dimorphic. Furthermore, these results show that blocking hypertrophy by active GSK-3β in this HCM model is not therapeutic.
Alterations in cerebral autoregulation and cerebral blood flow velocity during acute hypoxia: rest and exercise
Authors: Ainslie Philip N. , Barach Alice , Murrell Carissa , Hamlin Mike , Hellemans John , Ogoh Shigehiko
Published: September 2006

We examined the relationship between changes in cardiorespiratory and cerebrovascular function in 14 healthy volunteers with and without hypoxia [arterial O2 saturation (SaO2) ∼80%] at rest and during 60–70% maximal oxygen uptake steady-state cycling exercise. During all procedures, ventilation, end-tidal gases, heart rate (HR), arterial blood pressure (BP; Finometer) cardiac output (Modelflow), muscle and cerebral oxygenation (near-infrared spectroscopy), and middle cerebral artery blood flow velocity (MCAV; transcranial Doppler ultrasound) were measured continuously. The effect of hypoxia on dynamic cerebral autoregulation was assessed with transfer function gain and phase shift in mean BP and MCAV. At rest, hypoxia resulted in increases in ventilation, progressive hypocapnia, and general sympathoexcitation (i.e., elevated HR and cardiac output); these responses were more marked during hypoxic exercise ( P < 0.05 vs. rest) and were also reflected in elevation of the slopes of the linear regressions of ventilation, HR, and cardiac output with SaO2 ( P < 0.05 vs. rest). MCAV was maintained during hypoxic exercise, despite marked hypocapnia (44.1 ± 2.9 to 36.3 ± 4.2 Torr; P < 0.05). Conversely, hypoxia both at rest and during exercise decreased cerebral oxygenation compared with muscle. The low-frequency phase between MCAV and mean BP was lowered during hypoxic exercise, indicating impairment in cerebral autoregulation. These data indicate that increases in cerebral neurogenic activity and/or sympathoexcitation during hypoxic exercise can potentially outbalance the hypocapnia-induced lowering of MCAV. Despite maintaining MCAV, such hypoxic exercise can potentially compromise cerebral autoregulation and oxygenation.
A simplified two-component model of blood pressure fluctuation
Authors: Brychta Robert J. , Shiavi Richard , Robertson David , Biaggioni Italo , Diedrich André
Published: September 2006

We propose a simple moving-average (MA) model that uses the low-frequency (LF) component of the peroneal muscle sympathetic nerve spike rate (LFspike rate) and the high-frequency (HF) component of respiration (HFResp) to describe the LF neurovascular fluctuations and the HF mechanical oscillations in systolic blood pressure (SBP), respectively. This method was validated by data from eight healthy subjects (23–47 yr old, 6 male, 2 female) during a graded tilt (15° increments every 5 min to a 60° angle). The LF component of SBP (LFSBP) had a strong baroreflex-mediated feedback correlation with LFspike rate( r = −0.69 ± 0.05) and also a strong feedforward relation to LFspike rate[ r = 0.58 ± 0.03 with LFSBPdelay (τ) = 5.625 ± 0.15 s]. The HF components of spike rate (HFspike rate) and SBP (HFSBP) were not significantly correlated. Conversely, HFRespand HFSBPwere highly correlated ( r = −0.79 ± 0.04), whereas LFRespand LFSBPwere significantly less correlated ( r = 0.45 ± 0.08). The mean correlation coefficients between the measured and model-predicted LFSBP( r = 0.74 ± 0.03) in the supine position did not change significantly during tilt. The mean correlation between the measured and model-predicted HFSBPwas 0.89 ± 0.02 in the supine position. R2values for the regression analysis of the model-predicted and measured LF and HF powers indicate that 78 and 91% of the variability in power can be explained by the linear relation of LFspike rateto LFSBPand HFRespto HFSBP. We report a simple two-component model using neural sympathetic and mechanical respiratory inputs that can explain the majority of blood pressure fluctuation at rest and during orthostatic stress in healthy subjects.
Intracellular calcium handling in ventricular myocytes from mdx mice
Authors: Williams Iwan A. , Allen David G.
Published: September 2006

Duchenne muscular dystrophy (DMD) is a lethal degenerative disease of skeletal muscle, characterized by the absence of the cytoskeletal protein dystrophin. Some DMD patients show a dilated cardiomyopathy leading to heart failure. This study explores the possibility that dystrophin is involved in the regulation of a stretch-activated channel (SAC), which in the absence of dystrophin has increased activity and allows greater Ca2+ into cardiomyocytes. Because cardiac failure only appears late in the progression of DMD, we examined age-related effects in the mdx mouse, an animal model of DMD. Ca2+ measurements using a fluorescent Ca2+-sensitive dye fluo-4 were performed on single ventricular myocytes from mdx and wild-type mice. Immunoblotting and immunohistochemistry were performed on whole hearts to determine expression levels of key proteins involved in excitation-contraction coupling. Old mdx mice had raised resting intracellular Ca2+ concentration ([Ca2+]i). Isolated ventricular myocytes from young and old mdx mice displayed abnormal Ca2+ transients, increased protein expression of the ryanodine receptor, and decreased protein expression of serine-16-phosphorylated phospholamban. Caffeine-induced Ca2+ transients showed that the Na+/Ca2+ exchanger function was increased in old mdx mice. Two SAC inhibitors streptomycin and GsMTx-4 both reduced resting [Ca2+]i in old mdx mice, suggesting that SACs may be involved in the Ca2+-handling abnormalities in these animals. This finding was supported by immunoblotting data, which demonstrated that old mdx mice had increased protein expression of canonical transient receptor potential channel 1, a likely candidate protein for SACs. SACs may play a role in the pathogenesis of the heart failure associated with DMD. Early in the disease process and before the onset of clinical symptoms increased, SAC activity may underlie the abnormal Ca2+ handling in young mdx mice.
Thin-fiber mechanoreceptors reflexly increase renal sympathetic nerve activity during static contraction
Authors: Kim Jong Kyung , Hayes Shawn G. , Kindig Angela E. , Kaufman Marc P.
Published: September 2006

The renal vasoconstriction induced by the sympathetic outflow during exercise serves to direct blood flow from the kidney toward the exercising muscles. The renal circulation seems to be particularly important in this regard, because it receives a substantial part of the cardiac output, which in resting humans has been estimated to be 20%. The role of group III mechanoreceptors in causing the reflex renal sympathetic response to static contraction remains an open question. To shed some light on this question, we recorded the renal sympathetic nerve responses to static contraction before and after injection of gadolinium into the arterial supply of the statically contracting triceps surae muscles of decerebrate unanesthetized and chloralose-anesthetized cats. Gadolinium has been shown to be a selective blocker of mechanogated channels in thin-fiber muscle afferents, which comprise the afferent arm of the exercise pressor reflex arc. In decerebrate ( n = 15) and chloralose-anesthetized ( n = 12) cats, we found that gadolinium (10 mM; 1 ml) significantly attenuated the renal sympathetic nerve and pressor responses to static contraction (60 s) after a latent period of 60 min; both responses recovered after a latent period of 120 min. We conclude that thin-fiber mechanoreceptors supplying contracting muscle are involved in some of the renal vasoconstriction evoked by the exercise pressor reflex.
TRPC3 channels colocalize with Na+/Ca2+exchanger and Na+pump in axial component of transverse-axial tubular system of rat ventricle
Authors: Goel Monu , Zuo Cheng-Di , Sinkins William G. , Schilling William P.
Published: September 2006

Transient receptor potential canonical (TRPC) proteins form Ca2+-permeable, nonselective cation channels activated after stimulation of G protein-coupled membrane receptors linked to phospholipase C (PLC). Although the PLC/inositol phosphate signaling pathway is known to exist in heart, expression and subcellular distribution of TRPC channel proteins in ventricular myocardium have not been evaluated. Of the six members of the TRPC channel family examined here, only TRPC3 was found by Western blot analysis of membrane proteins from rodent or canine ventricle. Likewise, only TRPC3 was observed in immunofluorescence analysis of thin sections from rat ventricle. TRPC3 was also the only family member observed in neonatal rat ventricular myocytes in culture. In longitudinal sections of rat ventricle, TRPC3 was predominantly localized to the intercalated disk region of the myocyte. However, transverse sections through heart muscle or single isolated adult myocytes revealed TRPC3-specific labeling in a vast network of intracellular membranes, where it colocalized with the Na+-K+-ATPase (NKA) pump and the Na+/Ca2+exchanger (NCX) but not with the ryanodine receptor or the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump. Reciprocal immunoprecipitation assays from rat or canine ventricle showed that TRPC3 associates with NKA and NCX but not with the plasmalemmal Ca2+-ATPase pump. Immunoprecipitations from Sf9 insect cells heterologously expressing TRPC3, NKA, and NCX in various combinations revealed that NKA and NCX interact and that TRPC3 and NCX interact, but that TRPC3 does not directly associate with NKA. Together, these results suggest that TRPC3 is localized in the ventricular myocyte to the axial component of the transverse-axial tubular system, where it exists in a signaling complex that includes NCX and NKA.
Hemodynamics in the mouse aortic arch as assessed by MRI, ultrasound, and numerical modeling
Authors: Feintuch Akiva , Ruengsakulrach Permyos , Lin Amy , Zhang Ji , Zhou Yu-Qing , Bishop Jonathon , Davidson Lorinda , Courtman David , Foster F. Stuart , Steinman David A. , Henkelman R. Mark , Ethier C. Ross
Published: September 2006

Mice are widely used to study arterial disease in humans, and the pathogenesis of arterial diseases is known to be strongly influenced by hemodynamic factors. It is, therefore, of interest to characterize the hemodynamic environment in the mouse arterial tree. Previous measurements have suggested that many relevant hemodynamic variables are similar between the mouse and the human. Here we use a combination of Doppler ultrasound and MRI measurements, coupled with numerical modeling techniques, to characterize the hemodynamic environment in the mouse aortic arch at high spatial resolution. We find that the hemodynamically induced stresses on arterial endothelial cells are much larger in magnitude and more spatially uniform in the mouse than in the human, an effect that can be explained by fluid mechanical scaling principles. This surprising finding seems to be at variance with currently accepted models of the role of hemodynamics in atherogenesis and the known distribution of atheromatous lesions in mice.
Red blood cells prevent inhibition of hypoxic pulmonary vasoconstriction by nitrite in isolated, perfused rat lungs
Authors: Deem Steven , Min Jin-Hye , Moulding Jennifer D. , Eveland Randy , Swenson Erik R.
Published: September 2006

Nitrite reduction to nitric oxide (NO) may be potentiated by a nitrite reductase activity of deoxyHb and contribute to systemic hypoxic vasodilation. The effect of nitrite on the pulmonary circulation has not been well characterized. We explored the effect of nitrite on hypoxic pulmonary vasoconstriction (HPV) and the role of the red blood cell (RBC) in nitrite reduction and nitrite-mediated vasodilation. As to method, isolated rat lungs were perfused with buffer, or buffer with RBCs, and subjected to repeated hypoxic challenges, with or without nitrite. As a result, in buffer-perfused lungs, HPV was reduced at nitrite concentrations of 7 μM and above. Nitrite inhibition of HPV was prevented by excess free Hb and RBCs, suggesting that vasodilation was mediated by free NO. Nitrite-inhibition of HPV was not potentiated by mild acidosis (pH = 7.2) or xanthine oxidase activity. RBCs at 15% but not 1% hematocrit prevented inhibition of HPV by nitrite (maximum nitrite concentration of ∼35 μM) independent of perfusate Po2. Degradation of nitrite was accelerated by hypoxia in the presence of RBCs but not during buffer perfusion. In conclusion, low micromolar concentrations of nitrite inhibit HPV in buffer-perfused lungs and when RBC concentration is subphysiological. This effect is lost when RBC concentration approaches physiological levels, despite enhanced nitrite degradation in the presence of RBCs. These data suggest that, although deoxyHb may generate NO from nitrite, insufficient NO escapes the RBC to cause vasodilation in the pulmonary circulation under the dynamic conditions of blood flow through the lungs and that RBCs are net scavengers of NO.
Negative regulation of vascular smooth muscle cell migration by blood shear stress
Authors: Goldman Jeremy , Zhong Lin , Liu Shu Q.
Published: September 2006

Vortex blood flow with reduced blood shear stress in a vein graft has been hypothesized to promote smooth muscle cell (SMC) migration and intimal hyperplasia, pathological events leading to vein graft restenosis. To demonstrate that blood shear stress regulates these processes, we developed a modified vein graft model where the SMC response to reduced vortex blood flow was compared with that of control vein grafts. Vortex blood flow induced SMC migration and neointimal hyperplasia in control vein grafts, whereas reduction of vortex blood flow in the modified vein graft strongly suppressed these effects. A venous polymer implant with known fluid shear stress was employed to clarify the molecular mechanism of shear-dependent SMC migration in vivo. In the polymer implant, the phosphorylation of extracellular signal-regulated kinase (ERK1/2) and myosin light chain kinase (MLCK), found primarily in SMCs, increased from day 3 to day 5 and returned toward the control level from day 5 to day 10, with the peak phosphorylation associated with the maximal speed of SMC migration. Treatment with PD-98059 (an inhibitor specific to the ERK1/2 activator MEK1/2) significantly suppressed the phosphorylation of MLCK, suggesting a role for ERK1/2 in regulating the activity of MLCK. Treatment with PD-98059 or ML-7 (an inhibitor specific to MLCK) reduced shear stress-dependent SMC migration, resulting in an SMC distribution independent of fluid shear stress. These results suggest that fluid shear stress regulates SMC migration via the mediation of ERK1/2 and MLCK.
Restoration of diabetes-induced abnormal local Ca2+release in cardiomyocytes by angiotensin II receptor blockade
Authors: Yaras Nazmi , Bilginoglu Ayca , Vassort Guy , Turan Belma
Published: September 2006

Stimulation of local renin-angiotensin system and increased levels of oxidants characterize the diabetic heart. Downregulation of ANG II type 1 receptors (AT1) and enhancement in PKC activity in the heart point out the role of AT1blockers in diabetes. The purpose of this study was to evaluate a potential role of an AT1blocker, candesartan, on abnormal Ca2+release mechanisms and its relationship with PKC in the cardiomyocytes from streptozotocin-induced diabetic rats. Cardiomyocytes were isolated enzymatically and then incubated with either candesartan or a nonspecific PKC inhibitor bisindolylmaleimide I (BIM) for 6–8 h at 37°C. Both candesartan and BIM applied on diabetic cardiomyocytes significantly restored the altered kinetic parameters of Ca2+transients, as well as depressed Ca2+loading of sarcoplasmic reticulum, basal Ca2+level, and spatiotemporal properties of the Ca2+sparks. In addition, candesartan and BIM significantly antagonized the hyperphosphorylation of cardiac ryanodine receptor (RyR2) and restored the depleted protein levels of both RyR2 and FK506 binding protein 12.6 (FKBP12.6). Furthermore, candesartan and BIM also reduced the increased PKC levels and oxidized protein thiol level in membrane fraction of diabetic rat cardiomyocytes. Taken together, these data demonstrate that AT1receptor blockade protects cardiomyocytes from development of cellular alterations typically associated with Ca2+release mechanisms in diabetes mellitus. Prevention of these alterations by candesartan may present a useful pharmacological strategy for the treatment of diabetic cardiomyopathy.
Transcoronary gene transfer of SERCA2a increases coronary blood flow and decreases cardiomyocyte size in a Type 2 diabetic rat model
Authors: Sakata Susumu , Lebeche Djamel , Sakata Yuri , Sakata Naoya , Chemaly Elie R. , Liang LiFan , Nakajima-Takenaka Chikako , Tsuji Tsuyoshi , Konishi Noboru , del Monte Federica , Hajjar Roger J. , Takaki Miyako
Published: September 2006

The Otsuka Long-Evans Tokushima fatty rat is an animal model of Type 2 diabetes mellitus (DM), which is characterized by diastolic dysfunction associated with decreased sarcoplasmic reticulum Ca2+-ATPase (SERCA2a). The aim of this study was to examine whether gene transfer of SERCA2a can influence coronary blood flow and cardiomyocyte diameter in this model. DM rats were injected with adenovirus carrying SERCA2a (DM+SERCA) or β-galactosidase gene (DM+βGal). Coronary blood flow was measured in cross-circulated excised hearts 3 days after infection. Although in all groups coronary blood flow remained unchanged even if left ventricular (LV) volume or intracoronary Ca2+ infusion was increased, the DM+SERCA group showed a sustained increase in coronary blood flow compared with the other groups. This result suggests that the sustained high coronary blood flow is a specific response in SERCA2a-overexpressed hearts. Although the LV weight-to-body weight ratio (LV/BW) and cardiomyocyte diameter were higher in the DM and DM+βGal groups than in the non-DM group, in the DM+SERCA group, these measurements were restored to non-DM size. The percentages of collagen area in the three DM groups was significantly higher than results shown in non-DM rats, and there were no significant differences in collagen area percentage among the three DM groups. These results suggest that a lowered LV/BW by SERCA2a overexpression is due mainly to reduced size of cardiomyocytes without any changes in collagen area percentage. In conclusion, in DM failing hearts, SERCA2a gene transfer can increase coronary blood flow and reduce cardiomyocyte size without reduction in collagen production.
P2X receptor-mediated muscle pressor reflex in myocardial infarction
Authors: Gao Zhaohui , Xing Jihong , Sinoway Lawrence , Li Jianhua
Published: September 2006

A previous report from this laboratory demonstrated that the ATP-sensitive P2X receptor-mediated muscle pressor reflex was augmented in rats with heart failure (HF). The purpose of this study was to better understand the underlying mechanisms for this greater response in HF rats. We examined 1) responsiveness of the P2X receptor to α,β-methylene ATP (α,β-me-ATP), a P2X receptor agonist, in control and HF rats induced by myocardial infarction (MI); 2) the relationship between P2X-induced blood pressure response and left ventricular (LV) function; and 3) the expression of P2X receptors in the dorsal root ganglion (DRG) of control rats and rats with HF. Eight to 14 wk after coronary artery ligation, the severity of the MI was determined by echocardiography. In the first group of the experiment, α,β-me-ATP (0.0625, 0.125, 0.25, and 0.5 mM) was injected into the arterial blood supply of the hindlimb muscles to evoke a pressor response in 17 decerebrated rats (6 controls, 6 small MIs with infarcts of the LV between 10 and 35%, and 5 large MIs with infarcts >35%). The P2X agonist increased blood pressure, and the effect was significantly accentuated in large MI rats compared with small MI rats and control rats. A significant correlation was observed between α,β-me-ATP-evoked pressor response and the LV fractional shortening, an index of LV function. In the second group of the experiment, immunocytochemistry was used to examine the immunoreactivity of P2X receptor in the DRG neurons of small diameter fibers in six healthy control rats, five small MI, and five large MI rats. The percentage of P2X immunostaining-positive neurons in the DRG was markedly greater in large MI rats (52% vs. 29% in controls and 34% in small MIs, P < 0.05). In conclusion, our findings demonstrate that 1) muscle afferent-mediated pressor response of P2X activation was exaggerated in MI animals, and the responsiveness was related to the degree of LV dysfunction; and 2) augmented reflex response was associated with upregulated P2X receptors in the DRG neurons of thin fiber afferent nerves following MI. The data suggest that P2X-mediated responsiveness in the processing of muscle afferent signals may have important implications for understanding cardiovascular responses to exercise in HF.
Role of internalization of M2muscarinic receptor via clathrin-coated vesicles in desensitization of the muscarinic K+current in heart
Authors: Yamanushi T. T. , Shui Z. , Leach R. N. , Dobrzynski H. , Claydon T. W. , Boyett M. R.
Published: September 2006

In the heart, ACh activates the ACh-activated K+current ( IK,ACh) via the M2muscarinic receptor. The relationship between desensitization of IK,AChand internalization of the M2receptor has been studied in rat atrial cells. On application of the stable muscarinic agonist carbachol for 2 h, IK,AChdeclined by ∼62% with time constants of 1.5 and 26.9 min, whereas ∼83% of the M2receptor was internalized from the cell membrane with time constants of 2.9 and 51.6 min. Transfection of the cells with β-adrenergic receptor kinase 1 (G protein-receptor kinase 2) and β-arrestin 2 significantly increased IK,AChdesensitization and M2receptor internalization during a 3-min application of agonist. Internalized M2receptor in cells exposed to carbachol for 2 h was colocalized with clathrin and not caveolin. It is concluded that a G protein-receptor kinase 2- and β-arrestin 2-dependent internalization of the M2receptor into clathrin-coated vesicles could play a major role in IK,AChdesensitization.
Moxifloxacin but not ciprofloxacin or azithromycin selectively inhibits IL-8, IL-6, ERK1/2, JNK, and NF-κB activation in a cystic fibrosis epithelial cell line
Authors: Blau Hannah , Klein Keren , Shalit Itamar , Halperin Drora , Fabian Ina
Published: September 2006

Cystic fibrosis (CF) is associated with severe neutrophilic airway inflammation. We showed that moxifloxacin (MXF) inhibits IL-8 and MAPK activation in monocytic and respiratory epithelial cells. Azithromycin (AZM) and ciprofloxacin (CIP) are used clinically in CF. Thus we now examined effects of MXF, CIP, and AZM directly on CF cells. IB3, a CF bronchial cell line, and corrected C38 cells were treated with TNF-α, IL-1β, or LPS with or without 5–50 μg/ml MXF, CIP, or AZM. IL-6 and IL-8 secretion (ELISA), MAPKs ERK1/2, JNK, p38, and p65 NF-κB (Western blot) activation were measured. Baseline IL-6 was sixfold higher in IB3 than C38 cells but IL-8 was similar. TNF-α and IL-1β increased IL-6 and IL-8 12- to 67-fold with higher levels in IB3 than C38 cells post-TNF-α ( P < 0.05). Levels were unchanged following LPS. Baseline phosphorylated form of ERK1/2 (p-ERK1/2), JNK, and NF-κB p65 were higher in IB3 than C38 cells (5-, 1.4-, and 1.4-fold), and following TNF-α increased, as did the p-p38, by 1.6- to 2-fold. MXF (5–50 μg/ml) and CIP (50 μg/ml), but not AZM, suppressed IL-6 and IL-8 secretion by up to 69%. MXF inhibited TNF-α-stimulated MAPKs ERK1/2, 46-kDa JNK, and NF-κB up to 60%, 40%, and 40%, respectively. In contrast, MXF did not inhibit p38 activation, implying a highly selective pretranslational effect. In conclusion, TNF-α and IL-1β induce an exaggerated inflammatory response in CF airway cells, inhibited by MXF more than by CIP or AZM. Clinical trials are recommended to assess efficacy in CF and other chronic lung diseases.
Legionella pneumophila-induced PKCα-, MAPK-, and NF-κB-dependent COX-2 expression in human lung epithelium
Authors: N'Guessan Philippe Dje , Etouem Mirabelle O. , Schmeck Bernd , Hocke Andreas C. , Scharf Stefanie , Vardarova Kremena , Opitz Bastian , Flieger Antje , Suttorp Norbert , Hippenstiel Stefan
Published: September 2006

Legionella pneumophila causes community- and hospital-acquired pneumonia. Lung airway and alveolar epithelial cells comprise an important barrier against airborne pathogens. Cyclooxygenase (COX) and microsomal PGE2synthase-1 (mPGES-1)-derived prostaglandins like prostaglandin E2(PGE2) are considered as important regulators of lung function. Herein we tested the hypothesis that L. pneumophila induced COX-2 and mPGES-1-dependent PGE2production in pulmonary epithelial cells. Legionella induced the release of PGE2in primary human small airway epithelial cells and A549 cells. This was accompanied by an increased expression of COX-2 and mPGES-1 as well as an increased PLA2activity in infected cells. Deletion of the type IV secretion system Dot/Icm did not impair Legionella-related COX-2 expression or PGE2release in A549 cells. L. pneumophila induced the degradation of IκBα and activated NF-κB. Inhibition of IKK blocked L. pneumophila-induced PGE2release and COX-2 expression. We noted activation of p38 and p42/44 MAP kinase in Legionella-infected A549 cells. Moreover, membrane translocation and activation of PKCα was observed in infected cells. PKCα and p38 and p42/44 MAP kinase inhibitors reduced PGE2release and COX-2 expression. In summary, PKCα and p38 and p42/44 MAP kinase controlled COX-2 expression and subsequent PGE2release by Legionella-infected lung epithelial cells. These pathways may significantly contribute to the host response in Legionnaires' disease.
c-Src interacts with and phosphorylates RelA/p65 to promote thrombin-induced ICAM-1 expression in endothelial cells
Authors: Bijli Kaiser M. , Minhajuddin Mohd , Fazal Fabeha , O'Reilly Michael A. , Platanias Leonidas C. , Rahman Arshad
Published: September 2006

The procoagulant thrombin promotes polymorphonuclear leukocyte (PMN) adhesion to endothelial cells by a mechanism involving expression of intercellular adhesion molecule-1 (ICAM-1) via an NF-κB-dependent pathway. We now provide evidence that activation of c-Src is crucial in signaling thrombin-induced ICAM-1 expression via tyrosine phosphorylation of RelA/p65. Stimulation of human umbilical vein endothelial cells with thrombin resulted in a time-dependent activation of c-Src, with maximal activation occurring at 30 min after thrombin challenge. Inhibition of c-Src by pharmacological and genetic approaches impaired thrombin-induced NF-κB-dependent reporter activity and ICAM-1 expression. Analysis of the NF-κB pathway revealed that the effect of c-Src inhibition occurred independently of IκBα degradation and NF-κB DNA binding function and was not associated with exchange of NF-κB dimers. Phosphorylation of RelA/p65 at Ser536, an event mediating the transcriptional activity of DNA-bound RelA/p65, was also insensitive to c-Src inhibition. Interestingly, thrombin induced association of c-Src with RelA/p65, and inhibition of c-Src prevented this response, indicating that this interaction is contingent on activation of c-Src. We also observed that thrombin induced tyrosine phosphorylation of RelA/p65, and this phosphorylation was lost upon inhibition of c-Src, consistent with the requirement of activated c-Src for interaction with RelA/p65. These data implicate an important role of c-Src in phosphorylating RelA/p65 to promote the transcriptional activity of NF-κB and thereby ICAM-1 expression in endothelial cells.
Involvement of microtubules, p38, and Rho kinases pathway in 2-methoxyestradiol-induced lung vascular barrier dysfunction
Authors: Bogatcheva Natalia V. , Adyshev Djanybek , Mambetsariev Bolot , Moldobaeva Nurgul , Verin Alexander D.
Published: September 2006

2-Methoxyestradiol (2ME), a promising anti-tumor agent, is currently tested in phase I/II clinical trial to assess drug tolerance and clinical effects. 2ME is known to affect microtubule (MT) polymerization rather than act through estrogen receptors. We hypothesized that 2ME, similar to other MT inhibitors, disrupts endothelial barrier properties. We show that 2ME decreases transendothelial electrical resistance and increases FITC-dextran leakage across human pulmonary artery endothelial monolayer, which correlates with 2ME-induced MT depolymerization. Pretreatment of endothelium with MT stabilizer taxol significantly attenuates the decrease in transendothelial resistance. 2ME treatment results in the induction of F-actin stress fibers, accompanied by the increase in myosin light chain (MLC) phosphorylation. The experiments with Rho kinase (ROCK) and MLC kinase inhibitors and ROCK small interfering RNA (siRNA) revealed that increase in MLC phosphorylation is attributed to the ROCK activation rather than MLC kinase activation. 2ME induces significant ERK1/2, p38, and JNK phosphorylation and activation; however, only p38 activation is relevant to the 2ME-induced endothelial hyperpermeability. p38 activation is accompanied by a marked increase in MAPKAP2 and 27-kDa heat shock protein (HSP27) phosphorylation level. Taxol significantly decreases p38 phosphorylation and activation in response to 2ME stimulation. Vice versa, p38 inhibitor SB203580 attenuates MT rearrangement in 2ME-challenged cells. Together, these results indicate that 2ME-induced barrier disruption is governed by MT depolymerization and p38- and ROCK-dependent mechanisms. The fact that certain concentrations of 2ME induce endothelial hyperpermeability suggests that the issue of the maximum-tolerated dose of 2ME for cancer treatment should be addressed with caution.
Resealing of endothelial junctions by focal adhesion kinase
Authors: Quadri Sadiqa K. , Bhattacharya Jahar
Published: September 2006

Endothelial cell (EC) junctions determine vascular barrier properties and are subject to transient opening to allow liquid flux from blood to tissue. Although EC junctions open in the presence of permeability-enhancing factors, including oxidants, the mechanisms by which they reseal remain inadequately understood. To model opening and resealing of EC junctions in the presence of an oxidant, we quantified changes in H2O2-induced transendothelial resistance (TER) in monolayers of rat lung microvascular EC. During a 30-min exposure, H2O2(100 μM) decreased TER for an initial ∼10 min, indicating junctional opening. Subsequently, despite continuous presence of H2O2, TER recovered to baseline, indicating the activation of junctional resealing mechanisms. These bimodal TER transients matched the time course of loss and then gain of E-cadherin at EC junctions. The timing of the TER decrease matched the onset of focal adhesion formation, while F-actin increase at the cell periphery occurred with a time course that complemented the recovery of peripheral E-cadherin. In monolayers expressing a focal adhesion kinase (FAK) mutant (del-FAK) that inhibits FAK activity, the initial H2O2-induced junctional opening was present, although the subsequent junctional recovery was blocked. Expression of transfected E-cadherin was evident at the cell periphery of wild-type but not del-FAK-expressing EC. E-cadherin overexpression in del-FAK-expressing EC failed to effect major rescue of the junctional resealing response. These findings indicate that in oxidant-induced EC junction opening, FAK plays a critical role in remodeling the adherens junction to reseal the barrier.
Critical Roles for Non-pRb Targets of Human Papillomavirus Type 16 E7 in Cervical Carcinogenesis
Authors: Balsitis Scott , Dick Fred , Dyson Nicholas , Lambert Paul F.
Published: October 2006

High-risk human papillomaviruses (HPV) encode two oncogenes, E6 and E7, expressed in nearly all cervical cancers. In vivo, HPV-16 E7 has been shown to induce multiple phenotypes in the context of transgenic mice, including cervical cancer. E7 is a multifunctional protein known best for its ability to inactivate the tumor suppressor pRb. To determine the importance of pRb inactivation by E7 in cervical cancer, we pursued studies with genetically engineered mice. E7 expression in estrogen-treated murine cervix induced dysplasia and invasive cancers as reported previously, but targeted Rb inactivation in cervical epithelium was not sufficient to induce any cervical dysplasia or neoplasia. Furthermore, E7 induced cervical cancer formation even when the E7-pRb interaction was disrupted by the use of a knock-in mouse carrying an E7-resistant mutant Rb allele. pRb inactivation was necessary but not sufficient for E7 to overcome differentiation-induced or DNA damage–induced cell cycle arrest, and expression patterns of the E2F-responsive genes Mcm7 and cyclin E indicate that other E2F regulators besides pRb are important targets of E7. Together, these data indicate that non-pRb targets of E7 play critical roles in cervical carcinogenesis. (Cancer Res 2006; 12(18): 9393-400)
ERCC4 Associated with Breast Cancer Risk: A Two-Stage Case-Control Study Using High-throughput Genotyping
Authors: Milne Roger Laughlin , Ribas Gloria , González-Neira Anna , Fagerholm Rainer , Salas Antonio , González Emilio , Dopazo Joaquín , Nevanlinna Heli , Robledo Mercedes , Benítez Javier
Published: October 2006

The failure of linkage studies to identify further high-penetrance susceptibility genes for breast cancer points to a polygenic model, with more common variants having modest effects on risk, as the most likely candidate. We have carried out a two-stage case-control study in two European populations to identify low-penetrance genes for breast cancer using high-throughput genotyping. Single-nucleotide polymorphisms (SNPs) were selected across preselected cancer-related genes, choosing tagSNPs and functional variants where possible. In stage 1, genotype frequencies for 640 SNPs in 111 genes were compared between 864 breast cancer cases and 845 controls from the Spanish population. In stage 2, candidate SNPs identified in stage 1 (nominal P < 0.01) were tested in a Finnish series of 884 cases and 1,104 controls. Of the 10 candidate SNPs in seven genes identified in stage 1, one (rs744154) on intron 1 of ERCC4, a gene belonging to the nucleotide excision repair pathway, was associated with recessive protection from breast cancer after adjustment for multiple testing in stage 2 (odds ratio, 0.57; Bonferroni-adjusted P = 0.04). After considering potential functional SNPs in the region of high linkage disequilibrium that extends across the entire gene and upstream into the promoter region, we concluded that rs744154 itself could be causal. Although intronic, it is located on the first intron, in a region that is highly conserved across species, and could therefore be functionally important. This study suggests that common intronic variation in ERCC4 is associated with protection from breast cancer. (Cancer Res 2006; 66(19): 9420-7)