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<p>CD73 is a bifunctional glycosylphosphatidylinositol (GPI)-anchored membrane protein which functions as ecto-5′-nucleotidase and a membrane receptor for extracellular matrix protein (ECM). A large body of evidence demonstrates a critical involvement of altered purine metabolism and particularly, increased expression of CD73 in a number of human disorders, including cancer and immunodeficiency. Massive up-regulation of CD73 was also found in reactive astrocytes in several experimental models of human neuropathologies. In all the pathological contexts studied so far, the increased expression of CD73 has been associated with the altered ability of cells to adhere and/or migrate. Thus, we hypothesized that increased expression of CD73 in reactive astrocytes has a role in the process of astrocyte adhesion and migration. In the present study, the involvement of CD73 in astrocyte migration was investigated in the scratch wound assay (SW), using primary astrocyte culture prepared from neonatal rat cortex. The cultures were treated with one of the following pharmacological inhibitors which preferentially target individual functions of CD73: (a) α,β-methylene ADP (APCP), which inhibits the catalytic activity of CD73 (b) polyclonal anti-CD73 antibodies, which bind to the internal epitope of CD73 molecule and mask their surface exposure and (c) small interfering CD73-RNA (siCD73), which silences the expression of CD73 gene. It was concluded that approaches that reduce surface expression of CD73 increase migration velocity and promote wound closure in the scratch wound assay, while inhibition of the enzyme activity by APCP induces redistribution of CD73 molecules at the cell surface, thus indirectly affecting cell adhesion and migration. Application of anti-CD73 antibodies induces a decrease in CD73 activity and membrane expression, through CD73 molecules shedding and their release to the culture media. In addition, all applied pharmacological inhibitors differentially affect other aspects of astrocyte function in vitro, including reduced cell proliferation, altered expression of adenosine receptors and increased expression of ERK1/2. Altogether these data imply that CD73 participates in cell adhesion/migration and transmits extracellular signals through interactions with ECM.</p

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<p>CD73 is a bifunctional glycosylphosphatidylinositol (GPI)-anchored membrane protein which functions as ecto-5′-nucleotidase and a membrane receptor for extracellular matrix protein (ECM). A large body of evidence demonstrates a critical involvement of altered purine metabolism and particularly, increased expression of CD73 in a number of human disorders, including cancer and immunodeficiency. Massive up-regulation of CD73 was also found in reactive astrocytes in several experimental models of human neuropathologies. In all the pathological contexts studied so far, the increased expression of CD73 has been associated with the altered ability of cells to adhere and/or migrate. Thus, we hypothesized that increased expression of CD73 in reactive astrocytes has a role in the process of astrocyte adhesion and migration. In the present study, the involvement of CD73 in astrocyte migration was investigated in the scratch wound assay (SW), using primary astrocyte culture prepared from neonatal rat cortex. The cultures were treated with one of the following pharmacological inhibitors which preferentially target individual functions of CD73: (a) α,β-methylene ADP (APCP), which inhibits the catalytic activity of CD73 (b) polyclonal anti-CD73 antibodies, which bind to the internal epitope of CD73 molecule and mask their surface exposure and (c) small interfering CD73-RNA (siCD73), which silences the expression of CD73 gene. It was concluded that approaches that reduce surface expression of CD73 increase migration velocity and promote wound closure in the scratch wound assay, while inhibition of the enzyme activity by APCP induces redistribution of CD73 molecules at the cell surface, thus indirectly affecting cell adhesion and migration. Application of anti-CD73 antibodies induces a decrease in CD73 activity and membrane expression, through CD73 molecules shedding and their release to the culture media. In addition, all applied pharmacological inhibitors differentially affect other aspects of astrocyte function in vitro, including reduced cell proliferation, altered expression of adenosine receptors and increased expression of ERK1/2. Altogether these data imply that CD73 participates in cell adhesion/migration and transmits extracellular signals through interactions with ECM.</p

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<p>CD73 is a bifunctional glycosylphosphatidylinositol (GPI)-anchored membrane protein which functions as ecto-5′-nucleotidase and a membrane receptor for extracellular matrix protein (ECM). A large body of evidence demonstrates a critical involvement of altered purine metabolism and particularly, increased expression of CD73 in a number of human disorders, including cancer and immunodeficiency. Massive up-regulation of CD73 was also found in reactive astrocytes in several experimental models of human neuropathologies. In all the pathological contexts studied so far, the increased expression of CD73 has been associated with the altered ability of cells to adhere and/or migrate. Thus, we hypothesized that increased expression of CD73 in reactive astrocytes has a role in the process of astrocyte adhesion and migration. In the present study, the involvement of CD73 in astrocyte migration was investigated in the scratch wound assay (SW), using primary astrocyte culture prepared from neonatal rat cortex. The cultures were treated with one of the following pharmacological inhibitors which preferentially target individual functions of CD73: (a) α,β-methylene ADP (APCP), which inhibits the catalytic activity of CD73 (b) polyclonal anti-CD73 antibodies, which bind to the internal epitope of CD73 molecule and mask their surface exposure and (c) small interfering CD73-RNA (siCD73), which silences the expression of CD73 gene. It was concluded that approaches that reduce surface expression of CD73 increase migration velocity and promote wound closure in the scratch wound assay, while inhibition of the enzyme activity by APCP induces redistribution of CD73 molecules at the cell surface, thus indirectly affecting cell adhesion and migration. Application of anti-CD73 antibodies induces a decrease in CD73 activity and membrane expression, through CD73 molecules shedding and their release to the culture media. In addition, all applied pharmacological inhibitors differentially affect other aspects of astrocyte function in vitro, including reduced cell proliferation, altered expression of adenosine receptors and increased expression of ERK1/2. Altogether these data imply that CD73 participates in cell adhesion/migration and transmits extracellular signals through interactions with ECM.</p

Benfotiamine Attenuates Inflammatory Response in LPS Stimulated BV-2 Microglia

<div><p>Microglial cells are resident immune cells of the central nervous system (CNS), recognized as key elements in the regulation of neural homeostasis and the response to injury and repair. As excessive activation of microglia may lead to neurodegeneration, therapeutic strategies targeting its inhibition were shown to improve treatment of most neurodegenerative diseases. Benfotiamine is a synthetic vitamin B1 (thiamine) derivate exerting potentially anti-inflammatory effects. Despite the encouraging results regarding benfotiamine potential to alleviate diabetic microangiopathy, neuropathy and other oxidative stress-induced pathological conditions, its activities and cellular mechanisms during microglial activation have yet to be elucidated. In the present study, the anti-inflammatory effects of benfotiamine were investigated in lipopolysaccharide (LPS)-stimulated murine BV-2 microglia. We determined that benfotiamine remodels activated microglia to acquire the shape that is characteristic of non-stimulated BV-2 cells. In addition, benfotiamine significantly decreased production of pro-inflammatory mediators such as inducible form of nitric oxide synthase (iNOS) and NO; cyclooxygenase-2 (COX-2), heat-shock protein 70 (Hsp70), tumor necrosis factor alpha α (TNF-α), interleukin-6 (IL-6), whereas it increased anti-inflammatory interleukin-10 (IL-10) production in LPS stimulated BV-2 microglia. Moreover, benfotiamine suppressed the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and protein kinase B Akt/PKB. Treatment with specific inhibitors revealed that benfotiamine-mediated suppression of NO production was via JNK1/2 and Akt pathway, while the cytokine suppression includes ERK1/2, JNK1/2 and Akt pathways. Finally, the potentially protective effect is mediated by the suppression of translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the nucleus. Therefore, benfotiamine may have therapeutic potential for neurodegenerative diseases by inhibiting inflammatory mediators and enhancing anti-inflammatory factor production in activated microglia.</p></div

Functional characterization of benfotiamine effects in LPS-stimulated BV-2 microglia.

<p>(<b>A</b>) Real-time monitoring of BV-2 cell viability using xCELLigence RTCA analyzer. Representative graph showing the rate of proliferation in cells incubated in control medium (red line), medium with 1 μg/ml LPS (black line), or cells pretreated with benfotiamine, 50 μM (pink line), 100 μM (blue line) or 250 μM (green line) and then treated with LPS for 24 h. (<b>B</b>) Benfotiamine- induced alterations in cell morphology were analyzed using phase-contrast microscopy (left panels), whereas cell surface area was quantified by Phalloidin /Hoechst fluorescent staining (red/blue) microscopy (right panels), using AxioVisionRel 4.6 software. Insets: cell surface area was measured in five areas (138 × 104 μm²) per each cover-slip (n = 3) per experimental group in three independent experiments. (<b>C</b>) Bars present mean surface areas (± SEM) obtained from data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118372#pone.0118372.g001" target="_blank">Fig. 1B</a>. (<b>D</b>) Cell viability was assessed by crystal violet staining and results are displayed as percentage of control ± SEM (n = 3). *P < 0.05 control vs. LPS-induced BV-2 cells, # LPS vs. benfotiamine pretreated LPS activated BV-2 cells. Scale bar: 20 μm.</p

Effect of benfotiamine on cytokines expression and the release by BV-2 cells.

<p>Expression of TNF-α (A, B), IL-6 (C, D) and IL-10 (E, F) was analyzed at mRNA (A, C, E) and protein (B, D, F) level. Abundance of each mRNA transcript was expressed relative to GAPDH as internal control. Release of the cytokines was determined in the culture supernatants by ELISA. Bars represent mean ± SEM from <i>n</i> = 3 separate determinations. Significance levels shown inside the graphs: * - <i>p</i> < 0.05 control <i>vs</i>. LPS-induced BV-2 cells; # — LPS <i>vs</i>. benfotiamine pretreated LPS activated BV-2 cells.</p

Effect of pharmacological inhibitors on iNOS, TNF and IL6 gene expression followed by NO, IL-6 and TNF-α production.

<p>(<b>A, C, E</b>) Expression of iNOS, TNF and IL6 at mRNA level in BV-2 cells. Expression of iNOS, TNF and IL6-mRNA was assessed by RT-PCR, in control culture (white bar), LPS-treated culture (black bar), cultures pre-treated with U0126 (50 μM), SP600125 (20 μM) or LY294002 (20 μM) in presence or absence of benfotiamine (gray bars), 6 h following addition of LPS. iNOS, TNF and IL6-mRNA abundance was expressed relative to the abundance of GAPDH-mRNA, as an internal control. (<b>B, D, F</b>) The cultured supernatants were collected and analyzed for NO using Griess method, or TNF-α and IL6 production with ELISA. The data represent the mean ± SEM (n = 3), *P<0.05 control vs. LPS-induced BV-2 cells, # LPS vs. benfotiamine pretreated LPS activated BV-2 cells.</p

List of primers used for Real Time-PCR.

<p>List of primers used for Real Time-PCR.</p

The effect of benfotiamine on LPS—induced expression of proinflammatory effector molecules.

<p>(<b>A</b>) Expression of prostaglandin—endoperoxidase synthase 2 (PTGS2) at mRNA level in BV-2 cells. Expression of PTGS2-mRNA was assessed by RT-PCR, in control culture (white bar), LPS-treated culture (black bar) and cultures pre-treated with benfotiamine, 6 h following addition of LPS. PTGS2-mRNA abundance was expressed relative to the abundance of GAPDH-mRNA, as an internal control. (<b>B</b>) Expression of COX-2 at the protein level, determined by Western blot analysis. Bars show Cox-2/β-actin expression ratio relative to control (100%) ± SEM, from <i>n</i> = 3 separate determinations. Significance levels shown inside the graphs: *<i>p</i> < 0.05 control <i>vs</i>. LPS-induced BV-2 cells, # LPS <i>vs</i>. benfotiamine pretreated LPS activated BV-2 cells.</p

List of primary and secondary antibodies used for immunofluorescence (IF) and western blot (WB).

<p>List of primary and secondary antibodies used for immunofluorescence (IF) and western blot (WB).</p