Niacin Reverses Migratory Macrophage Foam Cell Arrest Mediated by oxLDL In Vitro

Introduction: Niacin reduces vascular oxidative stress and down regulates inducible nitric oxide synthase, an enzyme mediating proatherosclerotic effects in part by increasing oxidative stress. Here, we evaluate whether Niacin reverses the redox sensitive migratory arrest of macrophages in response to oxidised(ox) LDL uptake. Material and Methods: Migration of RAW264.7 cells, a murine macrophage cell line and bone marrow derived macrophages from wildtype and iNOS knockout mice was quantified using a modified Boyden chamber. Unstimulated cells or cells preincubated with oxLDL or non-oxidised (n)LDL were treated with Nicotinic acid or Nicotinamide. Nitric oxide, peroxynitrite and ROS production were assessed using electron paramagnetic resonance (ESR). Additionally, flow cytometry analysis of apoptosis, fokal adhesion kinase (FAK), phalloidin, CD36, F4/80 macrophage marker and iNOS gene expression (PCR) were assessed. Results: Migration of Nicotinic acid, Nicotinamide treated cells or unstimulated cells did not differ (P>0.05). oxLDL treatment significantly reduced migration vs. unstimulated cells (p, 0.05). In contrast, migratory arrest in response to oxLDL treatment was reversed by co-incubation with Nicotinic acid and Nicotinamide. The oxLDL-induced peroxynitrite formation in RAW264.7 cells was abolished by Niacin and glutathion (GSH) oxidation was significantly reduced. However, nitric oxide (NO)- and reactive oxygen species (ROS) production induced by oxLDL were not affected by Niacin treatment of RAW264.7 cells. In addition, Nicotinic acid and Nicotinamide reduced actin polymerization, a marker for migratory arrest. Discussion: Our data shows that oxLDL induced inhibition of macrophage migration in vitro can be reversed by Niacin. Furthermore, Niacin reduces peroxynitite formation and improves antioxidant GSH

Niacin Reverses Migratory Macrophage Foam Cell Arrest Mediated by oxLDL In Vitro

Introduction: Niacin reduces vascular oxidative stress and down regulates inducible nitric oxide synthase, an enzyme mediating proatherosclerotic effects in part by increasing oxidative stress. Here, we evaluate whether Niacin reverses the redox sensitive migratory arrest of macrophages in response to oxidised(ox) LDL uptake. Material and Methods: Migration of RAW264.7 cells, a murine macrophage cell line and bone marrow derived macrophages from wildtype and iNOS knockout mice was quantified using a modified Boyden chamber. Unstimulated cells or cells preincubated with oxLDL or non-oxidised (n)LDL were treated with Nicotinic acid or Nicotinamide. Nitric oxide, peroxynitrite and ROS production were assessed using electron paramagnetic resonance (ESR). Additionally, flow cytometry analysis of apoptosis, fokal adhesion kinase (FAK), phalloidin, CD36, F4/80 macrophage marker and iNOS gene expression (PCR) were assessed. Results: Migration of Nicotinic acid, Nicotinamide treated cells or unstimulated cells did not differ (P>0.05). oxLDL treatment significantly reduced migration vs. unstimulated cells (p, 0.05). In contrast, migratory arrest in response to oxLDL treatment was reversed by co-incubation with Nicotinic acid and Nicotinamide. The oxLDL-induced peroxynitrite formation in RAW264.7 cells was abolished by Niacin and glutathion (GSH) oxidation was significantly reduced. However, nitric oxide (NO)- and reactive oxygen species (ROS) production induced by oxLDL were not affected by Niacin treatment of RAW264.7 cells. In addition, Nicotinic acid and Nicotinamide reduced actin polymerization, a marker for migratory arrest. Discussion: Our data shows that oxLDL induced inhibition of macrophage migration in vitro can be reversed by Niacin. Furthermore, Niacin reduces peroxynitite formation and improves antioxidant GSH

Primers used for real-time PCR analysis.

<p>Primers used for real-time PCR analysis.</p

Migration assay of RAW264.7 cells.

<p><b>A</b>. Migrated RAW264.7 cells on the lower side of the membrane were stained with DAPI and counted under a fluorescence microscope using a ×10 objective. Representitive pictures of each treatment group. <b>B</b>. Migrated cells of each group. The migration of cells between each group was not significantly different. (P>0.05, n = 5). <b>C</b>. Migrated RAW264.7 cells on the lower side of the membrane were stained with DAPI and counted under a fluorescence microscope using a ×10 objective. Representitive pictures of each treatment group. <b>D</b>. Migrated cells of each group. Significance was determined by ANOVA. (*, p<0.05, Δ, p<0.05 compared with oxLDL group, n = 5).</p

Measurement of iNOS, p-FAK, 3-Nitrotyrosine expression, production of NO, ROS, superoxide, peroxynitrite and GSSG/GSH in RAW264.7 cells.

<p><b>A</b>. iNOS expression of RAW264.7 cells of each group was tested by “real time” PCR using comparative CT method. (*, P<0.05, n = 3). <b>B</b>. Representative western blot of iNOS expression of RAW264.7 cells of each group. (*, P<0.05, n = 3). <b>C</b>. The intracellular NO production was measured by ESR. (*, P<0.05, n = 5). <b>D</b>. Intracellular ROS and superoxide was measured by ESR. (*, P<0.05, n = 5). <b>E</b>. Intracellular ROS was measured by ESR. (*, P<0.05, n = 5). <b>F</b>. Representative western blot of 3-Nitrotyrosine. Band intensities were quantified by ImageJ and normalized to β-actin. (*, P<0.05, n = 3). <b>G</b>. Extracellular peroxynitrite was measured by ESR. (*, P<0.05, n = 6). <b>H</b>. GSSG/GSH of RAW264.7 cells measured by HPLC.(*, P<0.05, n = 4).</p

Migration assay of bone marrow derived macrophages.

<p><b>A</b>. Bone marrow derived macrophages were stained with fluorochrome labeled anti-F4/80 and anti-CD36 antibodies. Fluorescence intensity was analysed by flow cytometry. <b>B</b>. Migrated RAW264.7 cells on the lower side of the membrane were stained with DAPI and counted under a fluorescence microscope using a ×10 objective. Representitive pictures of each treatment group. <b>C</b>. Migration of oxLDL treated cells was significantly reduced compared to unstimulated cells. Migration of oxLDL treated cells was significantly different compared to Nicotinic acid 1 mM, Nicotinamide 1 mM and 3 mM. (*, P<0.05, n = 5).</p

Flow cytometry of Dil-oxLDL, and F4/80.

<p><b>A</b>. Cells were preincubated with Dil-oxLDL, then Nican in a concentration of 100 µM or 1 mM, or Nicotinamide in a concentration of 1 mM or 3 mM were added. The fluorescence intensity of Dil-oxLDL treated cells was significantly reduced when cell were treated with Nicotinamide at 3 mM. (*, P<0.05, n = 5). <b>B</b>. The fluorescence intensity of F4/80 of each group was not significantly different (P>0.05, n = 5). <b>C</b>. Fuorescence intensity of CD36 in each group. (*, P<0.05, n = 5).</p