Tag: AP48

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Differential Leukocyte and Endothelial Responses through VEGFR1 and VEGFR2

Purpose: Vascular endothelial growth factor (VEGF) induces angiogenesis and vascular permeability and is thought to be operative in several ocular vascular diseases. The VEGF isoforms are highly conserved among species; however, little is known about their differential biological functions in adult tissue. In the current study, the inflammatory potential of two prevalent VEGF isoform splice variants, VEGF120(121) and VEGF164(165), was studied in the transparent and avascular adult mouse cornea.

Methods: Controlled-release pellets containing equimolar amounts of VEGF120 and VEGF164 were implanted in corneas. The mechanisms underlying this differential response of VEGF isoforms were explored. The response of VEGF in cultured endothelial cells was determined by Western blot analysis. The response of VEGF isoforms in leukocytes was also investigated.

Results: VEGF164 was found to be significantly more potent at inducing inflammation. In vivo blockade of VEGF receptor (VEGFR)-1 significantly suppressed VEGF164-induced corneal inflammation. In vitro, VEGF165 more potently stimulated intracellular adhesion molecule (ICAM)-1 expression on endothelial cells, an effect that was mediated by VEGFR2. VEGF164 was also more potent at inducing the chemotaxis of monocytes, an effect that was mediated by VEGFR1. In an immortalized human leukocyte cell line, VEGF165 was found to induce tyrosine phosphorylation of VEGFR1 more efficiently.

conclusions. Taken together, these data identify VEGF164(165) as a proinflammatory isoform and identify multiple mechanisms underlying its proinflammatory biology.

http://www.iovs.org/content/45/2/368.long

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Angiopoietin-4 Inhibits Angiogenesis and Reduces Interstitial Fluid Pressure

Angiopoietins (Ang) are involved in the remodeling, maturation, and stabilization of the vascular network. Ang-4 was discovered more recently; thus, its effect on angiogenesis and its interplay with other angiogenic factors have not been equivocally established. The role of Ang-4 in angiogenesis was tested in Matrigel chambers implanted into the subcutaneous space of nude mice. Ang-4 inhibited the angiogenic response of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and GLC19 tumor cells. In Matrigel chambers with Ang-4-transfected cells, the mean response was significantly lower than that of mock cells. Subcutaneous tumor interstitial fluid pressure (IFP) was significantly lower in Ang-4-transfected GLC19 tumors than in mock-transfected tumors. IFP reduction in Ang-4-transfected tumors was comparable to the reduction seen after bevacizumab treatment. In vitro, we examined the effect of recombinant Ang-4 on endothelial cell migration in Boyden chambers. Human umbilical vein endothelial cell (HUVEC) migration induced by bFGF and VEGF was inhibited by Ang-4 to control levels. In conclusion, we show that rhAng-4, as well as transfection with Ang-4, inhibits angiogenesis induced by GLC19 tumor cells and that Ang-4 expression reduces elevated tumor IFP. In addition, we demonstrate that rhAng-4 inhibits HUVEC migration and growth factor-induced angiogenesis.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1592453/

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Cancer/Testis Antigen Expression in Human Mesenchymal Stem Cells: Down-regulation of SSX Impairs Cell Migration and Matrix Metalloproteinase 2 Expression

Several families of genes by and large located on the X chromosome encode proteins of unspecified function. Commonly known as cancer/testis (CT) antigens, they are considered, under normal conditions, only to be expressed in cells of the germ line and placenta. CT genes are also often expressed in cancer cells, hence their classification. Here we report that their expression in normal cells is wider spread and can be observed in cells with the potential for self-renewal and pleuripotency, namely, stem cells. Several CT genes and their products, CT antigens, including SSX, NY-ESO-1, and N-RAGE, were expressed in undifferentiated mesenchymal stem cells (MSCs) and down-regulated after osteocyte and adipocyte differentiation. To elucidate the possible overlapping function played by these genes in cancer and stem cells, a comparative analysis of the localization of their proteins was made. In addition, localization relative to other MSC markers was examined. This revealed that SSX localizes in the cytoplasm and overlap occurs in regions where matrix metalloproteinase 2 (MMP2) and vimentin accumulate. Nevertheless, it was found that no protein interactions between these molecules occur. Further investigation revealed that the migration of a melanoma cell line (DFW), which expresses SSX, MMP2, and vimentin, decreases when SSX is down-regulated. This decrease in cell migration was paralleled by a reduction in MMP2 levels. Analogous to this, SSX expression is down-regulated in MSCs after differentiation; concomitantly a reduction in MMP2 levels occurs. In addition, E-cadherin expression increases, mimicking a mesenchymal epithelial transition. These results afford SSX a functional role in normal stem cell migration and suggest a potentially similar function in cancer cell metastases.

http://cancerres.aacrjournals.org/content/65/6/2207.long

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Preferential chemotaxis of activated human CD4+ T cells by extracellular cyclophilin A

The recruitment and trafficking of leukocytes are essential aspects of the inflammatory process. Although chemokines are thought to be the main regulators of cell trafficking, extracellular cyclophilins have been shown recently to have potent chemoattracting properties for human leukocytes. Cyclophilins are secreted by a variety of cell types and are detected at high levels in tissues with ongoing inflammation. CD147 has been identified as the main signaling receptor for cyclophilin A (CypA) on human leukocytes. It is interesting that the expression of CD147 is elevated on leukocytes from inflamed tissue, suggesting a correlation among the presence of extracellular cyclophilins, CD147 expression, and inflammatory responses. Thus, cyclophilin-CD147 interactions may contribute directly to the recruitment of leukocytes into inflamed tissues. In the current studies, we show that activated human T lymphocytes express elevated levels of CD147, compared with resting T cells and that these activated T cells migrate more readily to CypA than resting cells. Furthermore, we show that unlike resting CD4+ T cells, the cyclophilin-mediated migration of activated T cells does not require interaction with heparan sulfate receptors but instead, is dependent on CD147 interaction alone. Such findings suggest that cyclophilin-CD147 interactions will be most potent when leukocytes are in an activated state, for example, during inflammatory responses. Thus, targeting cyclophilin-CD147 interactions may provide a novel approach for alleviating tissue inflammation.

http://www.jleukbio.org/content/82/3/613.long

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Localization of Rac2 via the C terminus and aspartic acid 150 specifies superoxide generation, actin polarity and chemotaxis in neutrophils

Despite having a high degree of sequence similarity, the Rho guanosine triphosphatases Rac1 and Rac2 regulate distinct functions in neutrophils. Here we demonstrate that the unique Rac2 localization and functions in neutrophils are regulated by two separate C-terminal motifs, the hypervariable domain and aspartic acid 150, one of which has not previously been linked to the function of Rho GTPases. In addition, we show an unexpected dependence of Rac1 localization on Rac2 activity in these same cells, demonstrating a degree of crosstalk between two closely related Rho GTPases. Thus, we have defined specific sequences in Rac that specify subcellular localization and determine the specificity of Rac2 in neutrophil chemotaxis and superoxide generation.

available by subscription at:

http://www.nature.com/ni/journal/v5/n7/full/ni1081.html

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Rapid densitometric determination of cell migration and cell adhesion in a microchemotaxis chamber

A new rapid staining and measuring method has been developed for the quantification of migrated cells in a microchemotaxis chamber. The migrated cells were, after staining, evaluated by a transmission densitometer. The method introduced here is more accurate and faster than those described previously. In addition the technique can be used to determine the adherent capacity of cells.

full text by subscription at:

http://www.sciencedirect.com/science/article/pii/0022175989900513

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Endothelial Responses to Oxidized Lipoproteins Determine Genetic Susceptibility to Atherosclerosis in Mice

Background—Oxidized LDL has been found within the subendothelial space, and it exhibits numerous atherogenic properties, including induction of inflammatory genes. We examined the possibility that variations in endothelial response to minimally modified LDL (MM-LDL) constitute one of the genetic components in atherosclerosis.

Methods and Results—By a novel explant technique, endothelial cells (ECs) were isolated from the aorta of inbred mouse strains with different susceptibilities to diet-induced atherosclerosis. Responses to MM-LDL were evaluated by examining the expression of inflammatory genes involved in atherosclerosis, including monocyte chemotactic protein-1 (MCP-1) and macrophage-colony–stimulating factor (M-CSF), an oxidative stress gene, heme oxygenase-1 (HO-1), and other, noninflammatory, genes. ECs from the susceptible mouse strain C57BL/6J exhibited dramatic induction of MCP-1, M-CSF, and HO-1, whereas ECs from the resistant strain C3H/HeJ showed little or no induction. In contrast, ECs from the 2 strains responded similarly to lipopolysaccharide.

Conclusions—These data provide strong evidence that genetic factors in atherosclerosis act at the level of the vessel wall.

http://circ.ahajournals.org/content/102/1/75.long

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Differential Effects of Two Fluorescent Probes on Macrophage Migration as Assessed by Manual and Automated Methods]

Fluorescent probes have been utilized to label leukocytes for both in vivo and in vitro studies of cell migration; however, the effects of such probes on migration have not been determined. The aim of this study was to examine the effects of two commonly used fluorescent probes on leukocyte chemotaxis. J774 macrophages were labeled with either calcein-acetoxymethyl ester (calcein-AM) or 2′,7′-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein, acetomethyl ester (BCECF-AM), then assayed for their ability to migrate to zymosan-activated serum (ZAS). Cell migration was quantified by two methods: visual counting of cells and measuring cell fluorescence. Using the cell counts, comparison of unlabeled and fluorescently labeled macrophages demonstrated that BCECF-AM decreased the number of cells responding to ZAS, while calcein-AM had essentially no effect. Neither probe significantly affected the number of cells migrating to medium alone. The inhibitory effects of BCECF-AM on cell migration increased with probe concentration (0.1-1.0 microM) and cell fluorescence. Cell viability was unaffected by either probe. In contrast to the results obtained by visual counting, measuring fluorescence of migrated cells did not reveal a significant difference between the chemotactic response of macrophages labeled with BCECF-AM and those labeled with calcein-AM. These experiments indicated that fluorescent probes can affect the chemotactic response and that inhibitory activity of these probes may not be detected when chemotaxis is quantified solely by automated methods.

pdf at: http://onlinelibrary.wiley.com/doi/10.1002/cyto.990190412/pdf

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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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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