bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sphingosine‐1‐phosphate induces COX‐2 expression via PI3K/Akt and p42/p44 MAPK pathways in rat vascular smooth muscle cells
Authors: Hsieh Hsi‐Lung , Wu Chou‐Bing , Sun Chi‐Chin , Liao Chang‐Hui , Lau Ying‐Tung , Yang Chuen‐Mao 
Published: February 2006

AbstractSphingosine 1‐phosphate (S1P) has been shown to regulate smooth muscle cell proliferation, migration, and vascular maturation. S1P increases the expression of several proteins including COX‐2 in vascular smooth muscle cells (VSMCs) and contributes to arteriosclerosis. However, the mechanisms regulating COX‐2 expression by S1P in VSMCs remain unclear. Western blotting and RT‐PCR analyses showed that S1P induced the expression of COX‐2 mRNA and protein in a time‐ and concentration‐dependent manner, which was attenuated by inhibitors of MEK1/2 (U0126) and PI3K (wortmannin), and transfection with dominant negative mutants of p42/p44 mitogen‐activated protein kinases (ERK2) or Akt. These results suggested that both p42/p44 MAPK and PI3K/Akt pathways participated in COX‐2 expression induced by S1P in VSMCs. In accordance with these findings, S1P stimulated phosphorylation of p42/p44 MAPK and Akt, which was attenuated by U0126, LY294002, or wortmannin, respectively. Furthermore, this up‐regulation of COX‐2 mRNA and protein was blocked by a selective NF‐κB inhibitor helenalin. Consistently, S1P‐stimulated translocation of NF‐κB into the nucleus was revealed by immnofluorescence staining. Moreover, S1P‐stimulated activation of NF‐κB promoter activity was blocked by phosphatidylinositol 3‐kinase (PI3K) inhibitor LY294002 and helenalin, but not by U0126, suggesting that involvement of PI3K/Akt in the activation of NF‐κB. COX‐2 promoter assay showed that S1P induced COX‐2 promoter activity mediated through p42/p44 MAPK, PI3K/Akt, and NF‐κB. These results suggested that in VSMCs, activation of p42/p44 MAPK, Akt and NF‐κB pathways was essential for S1P‐induced COX‐2 gene expression. Understanding the mechanisms involved in S1P‐induced COX‐2 expression on VSMCs may provide potential therapeutic targets in the treatment of arteriosclerosis. J. Cell. Physiol. © 2006 Wiley‐Liss, Inc.
Activation pathways of α4β1 integrin leading to distinct T‐cell cytoskeleton reorganization, Rac1 regulation and Pyk2 phosphorylation
Authors: Maqueda Alfredo , Moyano José V. , Gutiérrez‐López M. Dolores , Ovalle Susana , Rodríguez‐Frade José M. , Cabañas Carlos , Garcia‐Pardo Angeles 
Published: March 2006

Abstractα4β1 integrin is highly expressed in lymphocytes and is essential in hematopoiesis, extravasation, and the inflammatory response. α4β1 can be activated by intracellular signals elicited upon T‐cell activation by phorbol esters, CD3 crosslinking, or certain chemokine/receptor interactions (inside‐out activation). Divalent cations or certain anti‐ β1 mAbs (i.e., TS2/16) can also bind and activate integrins directly (outside‐in activation). In both cases, activation results in increased adhesion and/or affinity for ligands. It is not known if these various stimuli produce the same or different post‐adhesion events. To address this, we have studied the cytoskeleton organization and intracellular signaling following activation of α4β1 in Jurkat cells and in human T‐lymphoblasts. Treatment with Mn2+, α‐CD3 mAb or the chemokine SDF‐1α followed by attachment to the fibronectin fragment H89 or the endothelial molecule VCAM‐1 (α4β1 ligands), resulted in cell polarization and migration. In contrast, activation with PMA or TS2/16 induced cell spreading and strong adherence. Video microscopy and Transwell analyses confirmed these results, which correlated with different resistance to detachment under flow. Activation of the small GTPase RhoA or transfection with the constitutively active mutants V14RhoA or V12Rac1, abolished the α4β1‐induced cell polarization but did not affect cell spreading. Moreover, Rac1 activity was distinctly modulated by agents that induce a polarized or spread phenotype. The tyrosine kinase Pyk2 was highly phosphorylated upon induction of cell polarity but not during cell spreading. These results reveal novel properties of α4β1 integrin, namely the ability to trigger two types of T‐cell cytoskeletal response with different signaling requirements. J. Cell. Physiol. 207: 746–756, 2006. © 2006 Wiley‐Liss, Inc.
Heparanase is expressed in osteoblastic cells and stimulates bone formation and bone mass
Authors: Kram Vardit , Zcharia Eyal , Yacoby‐Zeevi Oron , Metzger Shula , Chajek‐Shaul Tova , Gabet Yankel , Müller Ralph , Vlodavsky Israel , Bab Itai 
Published: March 2006

AbstractHeparan sulfate proteoglycans (HSPGs) are ubiquitous macromolecules. In bone, they are associated with cell surfaces and the extracellular matrix (ECM). The heparan sulfate (HS) chains of HSPGs bind a multitude of bioactive molecules, thereby controlling normal and pathologic processes. The HS‐degrading endoglycosidase, heparanase, has been implicated in processes such as inflammation, vascularization associated with wound healing and malignancies, and cancer metastasis. Here we show progressive mRNA expression of the hpa gene (encoding heparanase) in murine bone marrow stromal cells undergoing osteoblastic (bone forming) differentiation and in primary calvarial osteoblasts. Bone marrow stromal cells derived from transgenic mice expressing recombinant human heparanase (rh‐heparanase) and MC3T3 E1 osteoblastic cells exposed to soluble rh‐heparanase spontaneously undergo osteogenic differentiation. In addition, the transgenic bone marrow stromal cells degrade HS chains. In wild‐type (WT) and hpa‐transgenic (hpa‐tg) mice, heparanase is weakly expressed throughout the bone marrow with a substantial increase in osteoblasts and osteocytes, especially in the hpa‐tg mice. Heparanase expression was absent in osteoclasts. Micro‐computed tomographic and histomorphometric skeletal analyses in male and female hpa‐tg versus WT mice show markedly increased trabecular bone mass, cortical thickness, and bone formation rate, but no difference in osteoclast number. Collectively, our data suggest that proteoglycans tonically suppress osteoblast function and that this inhibition is alleviated by HS degradation with heparanase. J. Cell. Physiol. © 2006 Wiley‐Liss, Inc.
Wound‐induced p38MAPK‐dependent histone H3 phosphorylation correlates with increased COX‐2 expression in enterocytes
Authors: Karrasch Thomas , Steinbrecher Kris A. , Allard Brigitte , Baldwin Albert S. , Jobin Christian 
Published: February 2006

AbstractGastrointestinal epithelial cell damage triggers an important biological response called restitution, a process aimed at re‐epithelializing the wounded areas. Unfortunately, little is known about the intrinsic molecular signaling events implicated in this host response. We hypothesized that wounding intestinal epithelial cells activates signaling pathways leading to chromatin modification and COX‐2 upregulation during restitution. Confluent rat IEC18 cells were mechanically wounded by multiple parallel scratches using a pipet tip. NF‐κB(Ser536), p38, and histone H3(Ser10) (H3S10) phosphorylation were determined by Western blot using specific phospho‐antibodies. COX‐2 gene expression was evaluated by RT‐PCR, Western Blot, and ELISA. Association of phosphorylated H3, RelA (NF‐κB), and RNA polymerase II to the COX‐2 gene promoter was evaluated by chromatin immunoprecipitation (ChIP). The specific inhibitors Bay11‐7082 and SB239063 as well as Ad5IκB‐superrepressor (Ad5IκBAA) and Ad5dnp38 were used to block NF‐κB‐ and p38‐signaling pathways, respectively. Wounding induced a rapid and sustained (24 h) phosphorylation of RelAS536, H3S10, and p38MAPK in enterocytes. ChIP analysis of the COX‐2 gene promoter demonstrated the presence of phospho‐H3S10 and recruitment of RelA and RNA polymerase II, a process blocked by SB239063. Finally, molecular blockade of NF‐κB (Ad5IκBAA) or p38MAPK (Ad5dnp38) signaling strongly inhibited enterocyte restitution. p38MAPK‐dependent histone 3 phosphorylation is an important component of the intestinal wound‐healing response. Targeting‐signaling pathways selectively involved in healing/restitution may provide a novel means to maintain or re‐establish host intestinal barrier integrity. J. Cell. Physiol. © 2006 Wiley‐Liss, Inc.
RNA interference reveals a differential role of FAK and Pyk2 in cell migration, leading edge formation and increase in focal adhesions induced by LPA in intestinal epithelial cells
Authors: Jiang Xiaohua , Jacamo Rodrigo , Zhukova Elena , Sinnett‐Smith James , Rozengurt Enrique 
Published: February 2006

AbstractIn the gastrointestinal mucosa, cell migration plays a crucial role in the organization and maintenance of tissue integrity but the mechanisms involved remain incompletely understood. Here, we used small‐interfering RNA (siRNA)‐mediated depletion of focal adhesion kinase (FAK) protein to determine the role of FAK in wound‐induced migration and cytoskeletal organization in the non‐transformed intestinal epithelial cells IEC‐6 and IEC‐18 stimulated with the G protein‐coupled receptors (GPCR) agonist lysophosphatidic acid (LPA). Treatment of these cells with FAK siRNA substantially reduced FAK expression, but did not affect the expression of proline‐rich tyrosine kinase 2 (Pyk2). Knockdown of FAK protein significantly inhibited LPA‐induced migration of both IEC‐18 and IEC‐6 cells. LPA induced reorganization of actin and microtubule cytoskeleton in the leading edge was largely inhibited in FAK siRNA‐transfected IEC‐18 cells. Interestingly, in contrast to the FAK−/− cells, which exhibit an increased number of prominent focal adhesions when plated on fibronectin, FAK knockdown IEC‐18 cells exhibited dramatically decreased number of focal adhesions in response to both LPA and fibronectin as compared with the control cells. We also used siRNAs to knockdown Pyk2 expression without reducing FAK expression. Depletion of Pyk2 did not prevent LPA‐induced migration or cytoskeletal reorganization in IEC‐18 cells. In conclusion, our study shows that FAK plays a critical role in LPA‐induced migration, cytoskeletal reorganization, and assembly of focal adhesions in intestinal epithelial cells whereas depletion of Pyk2 did not interfere with any of these responses elicited by LPA. J. Cell. Physiol. © 2006 Wiley‐Liss, Inc.
Mechanisms of AAV transduction in glaucoma‐associated human trabecular meshwork cells
Authors: Borrás Teresa , Xue Wei , Choi Vivian W. , Bartlett Jeffrey S. , Li Guorong , Samulski Richard J. , Chisolm Sarah S. 
Published: February 2006

AbstractBackgroundGlaucoma is a chronic eye disease which leads to irreversible blindness. The trabecular meshwork tissue controls intraocular pressure (IOP), which is the major risk factor for glaucoma. Gene therapy treatment of chronic diseases requires the use of long‐term expression, low toxicity and lack of immune response vectors. Adeno‐associated viruses (AAV) possess these characteristics but have been unable to transduce the trabecular meshwork. Because of the importance of regulating elevated IOP by long‐term gene therapy, we investigated mechanisms of AAV transduction to the human trabecular meshwork (TM).MethodsPrimary human trabecular meshwork cells (HTM) and perfused organ cultures were infected with rAAV2‐GFP, RGD‐pseudotyped rAAV2‐GFP alone, or combined with recombinant ΔE1/E3 adenoviruses. Intracellular rAAV2 DNA and RNA were measured by relative quantitative and real‐time TaqMan polymerase chain reaction (PCR). Host transcriptome was analyzed using high‐density oligonucleotide microarrays. One transduction mechanism was tested using self‐complementary AAV (scAAV).ResultsThe dramatic transduction enhancement obtained upon co‐infection of rAAV2 with ΔE1/E3 adenoviruses provides insights into transduction mechanisms in the HTM. Even if not transduced, rAAV2 enters TM cells. GeneChip analysis showed significant changes in host genes involved in cell cycle and DNA replication. Consequently, scAAV‐GFP transduction was highly efficient. Other transduction‐enhancement genes included coxsackie adenovirus receptor (CAR) and genes relevant to trabecular meshwork function.ConclusionsThe rate‐limiting step of AAV transduction was not viral entry failure but, at least in part, host downregulation of DNA replication. Additional specific host genes might be involved. Our study revealed genes and mechanisms which led for the first time to efficient AAV transduction of the HTM. Copyright © 2006 John Wiley & Sons, Ltd.
Radioprotective gene therapy through retroviral expression of manganese superoxide dismutase
Authors: Southgate Thomas D. , Sheard Victoria , Milsom Michael D. , Ward Timothy H. , Mairs Robert J. , Boyd Marie , Fairbairn Leslie J. 
Published: February 2006

AbstractBackgroundRadiotherapy for the control of cancer, either alone or in conjunction with chemotherapy, is often limited by normal tissue toxicity including haematopoietic toxicity. Exposure of cells to ionizing radiation leads to the formation of reactive oxygen species that are associated with radiation‐induced cytotoxicity. The antioxidant enzyme manganese superoxide dismutase (SOD2) catalyzes the dismutation of the superoxide anions into hydrogen peroxide.MethodsWe have investigated the potential of SOD2 overexpression, through retroviral gene transfer using a retrovirus optimized for transcription in early haematopoietic cells, to enhance the radioresistance of a human erythroleukaemic cell line and primary murine bone marrow. Using these as in vitro models we have investigated whether SOD2 gene therapy may be suitable for the protection of the haematopoietic compartment from the effects of ionizing radiation.ResultsHere we demonstrate using both biological and physical assays that overexpression of SOD2 protects haematopoietic cells from ionizing radiation injury. Our results show that an increase in the levels of SOD2 enzymatic activity within K562 cells (from 160.7 ± 23.6 to 321.8 ± 45.2 U/mg protein) or primary murine haematopoietic progenitor cells leads to both a significant decrease in DNA fragmentation and a significant increase in clonogenic survival, as evident by a significant increase in Dbar (from 2.66 to 3.42Gy), SF2 (from 0.52 to 0.73) values, and a significant decrease in the alpha value (from 0.3040 ± 0.037 to 0.0630 ± 0.037 Gy−1) when compared either to cells transduced with a retroviral vector encoding eGFP alone or to the parental line.ConclusionsThe results presented suggest that retroviral radioprotective gene therapy may be applicable to the haematopoietic compartment, enabling radiation dose escalation in cancer therapy. Copyright © 2006 John Wiley & Sons, Ltd.
Phage ϕC31 integrase‐mediated genomic integration of the common cytokine receptor gamma chain in human T‐cell lines
Authors: Ishikawa Yoshinori , Tanaka Nobuyuki , Murakami Kazuhiro , Uchiyama Toru , Kumaki Satoru , Tsuchiya Shigeru , Kugoh Hiroyuki , Oshimura Mitsuo , Calos Michele P. , Sugamura Kazuo 
Published: February 2006

AbstractBackgroundX‐linked severe combined immunodeficiency (SCID‐X1, X‐SCID) is a life‐threatening disease caused by a mutated common cytokine receptor γ chain (γc) gene. Although ex vivo gene therapy, i.e., transduction of the γc gene into autologous CD34+ cells, has been successful for treating SCID‐X1, the retrovirus vector‐mediated transfer allowed dysregulated integration, causing leukemias. Here, to explore an alternative gene transfer methodology that may offer less risk of insertional mutagenesis, we employed the ϕC31 integrase‐based integration system using human T‐cell lines, including the γc‐deficient ED40515(‐).MethodsA ϕC31 integrase and a neor gene expression plasmid containing the ϕC31 attB sequence were co‐delivered by electroporation into Jurkat cells. After G418 selection, integration site analyses were performed using linear amplification mediated‐polymerase chain reaction (LAM‐PCR). ED40515(‐) cells were also transfected with a γc expression plasmid containing attB, and the integration sites were determined. IL‐2 stimulation was used to assess the functionality of the transduced γc in an ED40515(‐)‐derived clone.ResultsFollowing co‐introduction of the ϕC31 integrase expression plasmid and the plasmid carrying attB, the efficiency of integration into the unmodified human genome was assessed. Several integration sites were characterized, including new integration sites in intergenic regions on chromosomes 13 and 18 that may be preferred in hematopoietic cells. An ED40515(‐) line bearing the integrated γc gene exhibited stable expression of the γc protein, with normal IL‐2 signaling, as assessed by STAT5 activation.ConclusionsThis study supports the possible future use of this ϕC31 integrase‐mediated genomic integration strategy as an alternative gene therapy approach for treating SCID‐X1. Copyright © 2006 John Wiley & Sons, Ltd.
EPI distortion correction from a simultaneously acquired distortion map using TRAIL
Authors: Priest Andrew N. , De Vita Enrico , Thomas David L. , Ordidge Roger J. 
Published: March 2006

AbstractPurposeTo develop a method for shot‐by‐shot distortion correction of single‐shot echo‐planar imaging (EPI) that is capable of correcting each image individually using a distortion measurement performed during acquisition of the image itself.Materials and MethodsThe recently‐introduced method known as two reduced acquisitions interleaved (TRAIL) was extended to measure the distribution of the main magnetic field B0 with each shot. This corresponded to a map of distortion, and allowed distortion to be corrected in the acquired images.ResultsDistortion‐corrected images were demonstrated in the human brain. The distortion field could be directly visualized using the “stripe” distribution imposed by the TRAIL pulse sequence. This confirmed the success of the correction. Over a time‐course measurement of 10 images, variance was reduced by using shot‐by‐shot distortion correction compared to correction with a constant field map.ConclusionShot‐by‐shot distortion correction may be performed for EPI images acquired using an extension of the TRAIL technique, ensuring that the correction reflects the actual distortion pattern and not merely a previously measured, but possibly no longer valid, distortion field. This avoids errors due to changes in the distortion field or misregistration of a previously measured distortion map resulting from subject motion. J. Magn. Reson. Imaging 2006. © 2006 Wiley‐Liss, Inc.
Myocardial tagging and strain analysis at 3 Tesla: Comparison with 1.5 Tesla imaging
Authors: Valeti V. Uma , Chun Wookjin , Potter Donald D. , Araoz Philip A. , McGee Kiaran P. , Glockner James F. , Christian Timothy F. 
Published: February 2006

AbstractPurposeTo determine whether imaging at 3 T could improve and prolong the tag contrast compared to images acquired at 1.5 T in normal volunteers, and whether such improvement would translate into the ability to perform strain measurements in diastole.Materials and MethodsNormal volunteers (N = 13) were scanned at 1.5 T (GE Signa CV/i) and 3.0 T (GE VH/i). An ECG‐triggered, segmented k‐space, spoiled‐gradient‐echo grid‐tagged sequence was used during cine acquisition. Tag contrast was determined by the difference of the mean signal intensity (SI) of the tagline to the mean SI of the myocardium divided by the standard deviation (SD) of the noise (CNRtag). Matched short‐axis (SA) slices were analyzed. Strain measurements were performed on images using a 2D strain analysis software program (harmonic phase (HARP)).ResultsThe average CNRtag over the cardiac cycle was superior at 3 T compared to 1.5 T for all slices (3 T: 23.4 ± 12.1, 1.5 T: 9.8 ± 8.4; P < 0.0001). This difference remained significant at cycle initiation, end‐systole, and the end R‐R interval (at cycle termination: 3 T = 14.0 ± 11.0 vs. 1.5 T = 4.4 ± 3.5; P < 0.01). Strain measures were obtainable only in early systole for 1.5 T images, but were robust throughout the entire R‐R interval for 3 T images.ConclusionImaging at 3 T had a significant benefit for myocardial tag persistence through the cardiac cycle. The improvement allowed strain analysis to be performed into diastole. J. Magn. Reson. Imaging 2006. © 2006 Wiley‐Liss, Inc.
In vivo porcine liver radiofrequency ablation with simultaneous MR temperature imaging
Authors: Vigen Karl K. , Jarrard Jerry , Rieke Viola , Frisoli Joan , Daniel Bruce L. , Pauly Kim Butts 
Published: February 2006

AbstractPurposeTo demonstrate in vivo MR‐guided temperature mapping during radiofrequency (RF) ablation of the liver with a commercially available RF generator modified to allow simultaneous RF treatment and MRI.Materials And MethodsA commercial RF generator was modified using passive filtering to allow the continuous application of the treatment current during MRI studies. A total of six ablations were performed with the device in vivo in three porcine livers, and imaging was concurrently performed using one of two different temperature mapping strategies.ResultsMR images acquired during RF ablation demonstrated no noticeable interference from the RF ablation device, which was operated at clinically relevant power levels. Temperature maps showed areas of heating that were consistent with the dimensions of the RF ablation probe, with some asymmetry (likely depending on the orientation of the probe and heat propagation effects), and some differences in heating‐spot area stability depending on the specific temperature mapping strategy used. Lesions were visualized on post‐ablation imaging and sectioning.ConclusionThe feasibility of performing RF ablation with a modified commercial RF generator simultaneously with MRI was demonstrated. Interference‐free MR temperature maps were produced with both variable respiratory motion and mechanical ventilation, and showed the extent of heating as the ablation progressed. J. Magn. Reson. Imaging 2006. © 2006 Wiley‐Liss, Inc.
Alterations of the proton‐T2 time in relaxed skeletal muscle induced by passive extremity flexions
Authors: Rump Jens , Braun Jürgen , Papazoglou Sebastian , Taupitz Matthias , Sack Ingolf 
Published: March 2006

AbstractPurposeTo demonstrate reciprocal changes of the apparent proton‐T2 time in the biceps and triceps due to passive contraction and extension of the muscle fibers.Materials and MethodsThe contraction state of the upper arm muscles of six healthy volunteers was passively changed by alternating the forearm position between the straight‐arm position and an elbow flexion of 90°. The relaxation of the muscle during passive contraction and extension was measured with the use of muscle electromyography (EMG) experiments. Spin‐echo (SE) MRI with increasing echo times (TEs) of 12–90 msec was used to acquire the averaged signal decay of the segmented biceps and triceps. The apparent T2 was deduced using monoexponential least‐square fitting.ResultsThe median T2 alterations in biceps and triceps among all volunteers were found to be 1.2 and –1.3 msec in the straight and bent forearm positions, respectively. The confidence intervals (0.5 to 1.7 msec in biceps, and –2.6 to –1.1 msec in triceps) clearly indicate that proton‐T2 in MR images is significantly (P < 0.05) prolonged with muscle contraction.ConclusionThe observed increase of the proton‐T2 time was correlated with a passive contraction of skeletal muscle fibers. This passive effect can be attributed to changes in the intracellular water mobility corresponding to the well‐known “active” T2 increase that occurs after stimulation of muscle. J. Magn. Reson. Imaging 2006. © 2006 Wiley‐Liss, Inc.