Category: Knowledgebase

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An improved method for the quantitation of cellular migration: Role of αvβ3 integrin in endothelial and smooth muscle cell migration

The present study was undertaken to develop a simple and improvedmethod for the accurate quantitation of cellular migration and to examinethe role of agrvbeta3 integrins in different cellular migration. Using our newly developed micro-volume chemotaxis assay, we developed an improved quantitative method to measure in vitro chemotaxis of smooth muscle or endothelial cells toward different extracellular matrix proteins. The convenience in setup and counting of migrated cells using this method allows for large capacity screening and for various research applications with other cells as well. The signal to noise ratios were in the range of 10/1, along with about 10–20% intra- or inter-assay variabilities. Using this method, we have determined that either vitronectin at 0.4 µg/well or osteopontin at 0.4 µg/well are selective agrvbeta3 chemoattractants for endothelial or smooth muscle cells (0.5 × 105 cells/well). Additionally, a selective agrvbeta3 small molecule peptiddomimetic, monoclonal antibody LM609, or an anti-beta3 (agrvbeta3/agrIIbeta3) anti-body, c7E3 demonstrated maximal inhibition of cellular migration toward vitronectin or osteopontin. These data suggest the potential utility of this method in assessing the role of various mechanisms in cellular migration and also suggests the potential implication of an agrvbeta3 antagonist in blocking pathological processes involving endothelial or smooth muscle cell adhesion/migration.

link to pdf at: http://www.springerlink.com/content/k60v0373k4962116/

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Retinoblastoma Protein Expression Leads to Reduced Oct-1 DNA Binding Activity and Enhances Interleukin-8 Expression

Tumor cell lines with a defective retinoblastoma gene are unableto transcribe the HLA class II genes in response to IFN-{gamma} treatment,and reconstitution of functional Rb rescues IFN-{gamma}-induced classII gene expression. However, the molecular mechanism of Rb rescueof the class II genes is unknown. We have examined the effectof Rb expression on the activation of the promoter for HLA-DRA,the prototype class II gene. Oct-1, a POU domain transcriptionfactor, was identified as a repressor of HLA-DRA promoter activityin the Rb-defective cells. Rb expression led to phosphorylationof Oct-1, thus relieving its repressive effect. Oct-1 has alsobeen shown to repress interleukin 8 promoter activity. Consistentwith reduced levels of Oct-1 DNA binding activity in the Rb-transformedcell lines, interleukin 8 expression is higher in these celllines.

http://cgd.aacrjournals.org/cgi/content/full/10/7/457

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HMG-CoA Reductase Inhibitors Prevent Migration of Human Coronary Smooth Muscle Cells Through Suppression of Increase in Oxidative Stress

In vitro and in vivo evidence of a decrease in vascular smooth muscle cell (SMC) migration induced by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors has been reported. When added to SMC cultures for 6 hours, the HMG-CoA reductase inhibitors fluvastatin, simvastatin, and pravastatin at 1 μmol/L resulted in a 48%, 50%, and 16% suppression, respectively, of human coronary SMC migration; these reductions mirrored the suppression in oxidative stress induced by 1 μmol/L lysophosphatidylcholine (lyso-PC) of 50%, 53% and 19%, respectively. The hydroxylated metabolites of fluvastatin, M2 and M3, at 1 μmol/L also suppressed the enhancement of SMC migration by 58% and 45% and the increase in oxidative stress induced by lyso-PC of 58% and 49%, respectively. Lyso-PC activated phospholipase D and protein kinase C (PKC), and this activation was also suppressed by HMG-CoA reductase inhibitors. The inhibition of phospholipase D and PKC was reversed by 100 μmol/L mevalonate, its isoprenoid derivative, farnesol, and geranylgeraniol but not by 10 μmol/L squalene. Antisense oligodeoxynucleotides at 5 μmol/L to PKC-α, but not those to the PKC-β isoform, suppressed the lyso-PC–mediated increases in SMC migration and oxidative stress. These findings suggest that HMG-CoA reductase inhibitors have direct antimigratory effects on the vascular wall beyond their effects on plasma lipids and that they might exert such antimigratory effects via suppression of the phospholipase D– and PKC (possibly PKC-α)-induced increase in oxidative stress, which might in turn prevent significant coronary artery disease.

http://atvb.ahajournals.org/content/21/6/937.long

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Cigarette Smoke Inhibits Human Bronchial Epithelial Cell Repair Processes

By interfering with the ability of airway epithelial cells to support repair processes, cigarette smoke could contribute to alterations of airway structures and functions that characterize chronic obstructive pulmonary disease (COPD). The current study assessed the ability of cigarette smoke extract (CSE) to alter human airway epithelial cell chemotaxis, proliferation, and contraction of three-dimensional collagen gels, a model of extracellular matrix remodeling. The volatile components contained in cigarette smoke, acetaldehyde and acrolein, were able to inhibit all three processes. Nonvolatile components contained within lyophilized CSE also inhibited chemotaxis but displayed no activity in the other two bioassays. CSE also inhibited the ability of airway epithelial cells to release transforming growth factor (TGF)- β and fibronectin. Exogenous fibronectin was unable to restore epithelial cell contraction of collagen gels. Exogenous TGF- β partially restored the ability of airway epithelial cells to contract collagen gels and to produce fibronectin. This supports a role for inhibition of TGF- β release in mediating the inhibitory effects of cigarette smoke. Taken together, the results of the current study suggest that epithelial cells present in the airways of smokers may be altered in their ability to support repair responses, which may contribute to architectural disruptions present in the airways in COPD associated with cigarette smoking.

http://ajrcmb.atsjournals.org/content/25/6/772.long

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Interferon-g Upregulates the c-Met/Hepatocyte Growth Factor Receptor Expression in Alveolar Epithelial Cells

In the repair process after lung injury, the regeneration of alveolar epithelial cells plays an important role by covering the damaged alveolar wall and preventing the activated fibroblasts from invading the intra- alveolar spaces. Hepatocyte growth factor (HGF) is a potent mitogen for alveolar epithelial cells and has been reported to be capable of repressing the fibrosing process by connecting to the c-Met/HGF receptor on alveolar epithelial cells. However, it has been reported that the c-Met expression was downregulated in an acute phase of lung injury, which may limit the effect of HGF for therapeutic use. In the present study we observed that interferon (IFN)-γ upregulates the c-Met messenger RNA (mRNA) and protein expression in A549 alveolar epithelial cells. We analyzed the mechanism of this upregulation and found that IFN-γ enhances the transcription of the c-met proto-oncogene, and that it does not prolong the stability of the c-Met mRNA. HGF is known to act as a motogen as well as a mitogen for epithelial cells. We also found that the migratory activity of A549 cells induced by HGF is strongly enhanced by preincubation with IFN-γ. Finally, we administered recombinant IFN-γ to C57BL/6 mice and confirmed that this upregulation is also observed in vivo. These results suggest that the combination of HGF and IFN-γ co

http://ajrcmb.atsjournals.org/content/21/4/490.long

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Exploitation of Interleukin-8-Induced Neutrophil Chemotaxis by the Agent of Human Granulocytic Ehrlichiosis

The agent of human granulocytic ehrlichiosis (HGE) is an obligate intracellular bacterium with a tropism for neutrophils; however, the mechanisms of bacterial dissemination are not yet understood. Interleukin-8 (IL-8) is a chemokine that induces neutrophil migration to sites of infection for host defense against pathogens. We now show that HGE bacteria, and the HGE-44 protein, induce IL-8 secretion in a promyelocytic (HL-60) cell line that has been differentiated along the neutrophil lineage with retinoic acid and in neutrophils. Infected HL-60 cells also demonstrate upregulation of CXCR2, an IL-8 receptor, but not CXCR1. Human neutrophils migrate towardsEhrlichia sp.-infected cells in a chemotaxis chamber assay, and this movement can be blocked with antibodies to IL-8. Finally, immunocompetent and severe combined immunodeficient mice administered CXCR2 antisera, and CXCR2−/− mice that lack the human IL-8 receptor homologue, are much less susceptible to granulocytic ehrlichiosis than are control animals. These results demonstrate that HGE bacteria induce IL-8 production by host cells and, paradoxically, appear to exploit this chemokine to enhance infection.

http://iai.asm.org/content/69/9/5577.long

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Critical role of endothelial CXCR2 in LPS-induced neutrophil migration into the lung

In models of acute lung injury, CXC chemokine receptor 2 (CXCR2) mediates migration of polymorphonuclear leukocytes (PMNs) into the lung. Since CXCR2 ligands, including CXCL1 and CXCL2/3, are chemotactic for PMNs, CXCR2 is thought to recruit PMNs by inducing chemotactic migration. In a model of PMN recruitment to the lung, aerosolized bacterial LPS inhalation induced PMN recruitment to the lung in wild-type mice, but not in littermate CXCR2–/– mice. Surprisingly, lethally irradiated wild-type mice reconstituted with CXCR2–/– BM still showed about 50% PMN recruitment into bronchoalveolar lavage fluid and into lung interstitium, but CXCR2–/– mice reconstituted with CXCR2–/– BM showed no PMN recruitment. Conversely, CXCR2–/– mice reconstituted with wild-type BM showed a surprisingly large defect in PMN recruitment, inconsistent with a role of CXCR2 on PMNs alone. Cell culture, immunohistochemistry, flow cytometry, and real-time RT-PCR were used to show expression of CXCR2 on pulmonary endothelial and bronchial epithelial cells. The LPS-induced increase in lung microvascular permeability as measured by Evans blue extravasation required CXCR2 on nonhematopoietic cells. Our data revealed what we believe to be a previously unrecognized role of endothelial and epithelial CXCR2 in LPS-induced PMN recruitment and lung injury.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1366502/

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CXC Chemokine Receptor 5 Expression Defines Follicular Homing T Cells with B Cell Helper Function

Leukocyte traffic through secondary lymphoid tissues is finely tuned by chemokines. We have studied the functional properties of a human T cell subset marked by the expression of CXC chemokine receptor 5 (CXCR5). Memory but not naive T cells from tonsils are CXCR5+ and migrate in response to the B cell–attracting chemokine 1 (BCA-1), which is selectively expressed by reticular cells and blood vessels within B cell follicles. Tonsillar CXCR5+ T cells do not respond to other chemokines present in secondary lymphoid tissues, including secondary lymphoid tissue chemokine (SLC), EBV-induced molecule 1 ligand chemokine (ELC), and stromal cell–derived factor 1 (SDF-1). The involvement of tonsillar CXCR5+ T cells in humoral immune responses is suggested by their localization in the mantle and light zone germinal centers of B cell follicles and by the concomitant expression of activation and costimulatory markers, including CD69, HLA-DR, and inducible costimulator (ICOS). Peripheral blood CXCR5+ T cells also belong to the CD4+ memory T cell subset but, in contrast to tonsillar cells, are in a resting state and migrate weakly to chemokines. CXCR5+ T cells are very inefficient in the production of cytokines but potently induce antibody production during coculture with B cells. These properties portray CXCR5+ T cells as a distinct memory T cell subset with B cell helper function, designated here as follicular B helper T cells (TFH).

http://jem.rupress.org/content/192/11/1553.long

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Behavior, reproduction, and immunity of crated pregnant gilts: Effects of high dietary fiber and rearing environment

The objective of this study was to examine effects of increased gut fill and diverse developing environments on pregnant gilts’ behavior and physiology. Gilts were cross-fostered at 1 d of age and transferred to either an indoor or outdoor production unit. Littermate gilts remained in their different environments during development and were moved into individual gestation crates in an indoor gestation unit. Of the 42 gilts, 19 were fed a control diet of fortified sorghum-soybean meal and 23 were fed the same diet with 25% beet pulp (high fiber). Control sows ate 2.0 kg/d and high-fiber sows ate 2.67 kg/d in a large pellet (thus resulting in approximately equal energy intake and differing total dietary intakes). Pregnant gilts had behavior and immune measures sampled at 30, 60, and 90 d of gestation. The day x diet interaction was significant (P = 0.01) for duration of standing: sows fed high-fiber diets stood less on d 30, but on d 60 and 90 they and the control sows stood for a similar duration. Sham chewing duration and frequency showed significant (P < 0.05) effects of gestation stage x diet x environment. Gilts reared outdoors and fed high fiber increased sham chewing over gestation, whereas all other treatment groups decreased this behavior over time. Outdoor-reared gilts had greater (P < 0.05) frequency and duration of drinking behavior than indoor-reared gilts. White blood cell numbers were higher (P < 0.05) for gilts fed high-fiber diets than for gilts fed the control diet. Immune (humoral and cellular systems) and reproductive measures (farrowing rate and litter size) and plasma cortisol concentrations were generally not influenced (P > 0.10) by diets and rearing environments, suggesting that in spite of significant changes in behavior and feed intake gilts’ immune systems were not suppressed or enhanced. Behavioral data alone suggested that indoor-reared gilts showed fewer behavioral adaptations to the crates than outdoor-reared gilts. However, immune measures did not indicate that any treatments resulted in physiological effects indicative of stress.

link to pdf at: http://www.journalofanimalscience.org/content/79/6/1466.long

 

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NADPH oxidase activity is required for endothelial cell proliferation and migration

NADPH oxidase has been shown to play an important role in cardiovascular biology. The goal of the present study was to determine whether NADPH oxidase activity is important for endothelial cell growth and migration. In proliferation assays, growth factor- or serum-induced DNA synthesis in three different types of human endothelial cells was abrogated by inhibitors of NADPH oxidase, but not by inhibitors of xanthine oxidase or nitric oxide synthase. Moreover, vascular endothelial growth factor-induced migration of human endothelial cells was suppressed in the presence of NADPH oxidase inhibitors. These results support a potential role for NADPH oxidase in mediating angiogenesis.

full text by subscription at:

http://linkinghub.elsevier.com/retrieve/pii/S0014-5793%2800%2902305-X