Effect of GPx-1 deficiency and MAPK signaling pathways inhibitors on macrophage proliferation.

<p>Macrophages were collected after differentiation of thioglycollate-elicited mouse peritoneal macrophages for 3 days in 10 ng/ml MCSF. <b>A left panel</b>, macrophages (2,5×10⁴ cells) were incubated with MCSF or oxLDL, respectively, and BrdU for another 48 hours. Subsequently, the proliferative activity was investigated with a BrdU-based chemiluminescence assay and expressed as relative proliferation rate relating to control cells from ApoE^−/− mice without stimulus. Data represent means ± SD of 4 to 5 separate experiments. *p<0.05 or **p<0,01 above the histogram indicate statistically significant differences between the different genotypes and below the histogram compared with cells without treatment of MCSF or oxLDL. <b>A right panel</b>, macrophages from GPx-1^−/−ApoE^−/− mice were pretreated with 10 µM ebselen for 1 h and incubated with 10 µg/ml oxLDL and BrdU for another 48 h. The proliferation rate was expressed as the relative proliferation rate relating to the proliferative activity of untreated cells. Data represent means ± SD of 3 independent experiments. <b>B,</b> macrophages of GPx-1^−/−ApoE^−/− (upper panel) and ApoE^−/− (lower panel) mice were pre-incubated with 75 µM PD98059 or 15 µM U0126, respectively, for 1 h and then incubated with 10 ng/ml MCSF (left) or 10 µg/ml oxLDL (right) and BrdU for another 48 h. The proliferation rate was expressed as the relative proliferation rate relating to the proliferative activity of untreated cells. Data represent means ± SD of 3 to 5 independent experiments. <b>C,</b> Representative immunohistochemical staining for the proliferation marker PCNA in atherosclerotic lesions of the aortic sinus demonstrating more pronounced positive nuclear staining in GPx-1^−/−ApoE^−/− mice (left panel) than ApoE^−/− mice (right panel). The vessel lumen is to the upper left-hand corner. The demarcation between intima and media is indicated by arrowheads.</p

Localization of GPx-1 in mice atherosclerotic lesions.

<p>GPx-1 mRNA and protein expression, macrophages and SMCs in sequential sections of the aortic sinus of ApoE^−/− (<b>A, C</b>) and GPx-1^−/−ApoE^−/− (<b>B</b>) mice. <b>A,</b> GPx-1 mRNA expression was detected by <i>in situ</i>-hybridization (upper panels) and both macrophages and SMCs were detected by immunohistochemistry (middle panels, see Methods). Upper left panel: anti-sense probe; upper right panel: corresponding section hybridized with the sense probe for GPx-1 (no signal). <b>B,</b> Control sections of GPx-1^−/−ApoE^−/− mice showed no expression of GPx-1 mRNA, neither with the anti-sense nor with the sense probe (upper panels). The representative atherosclerotic lesion containes both macrophages (lower left panel) and SMCs (lower right panel). <b>C,</b> Representative double immunohistochemical staining for GPx-1 (brown) and macrophages (red; left panel) and GPx-1 (brown) and SMCs (red; right panel) in ApoE^−/− mice. Note the close intermingling and overlapping of the different antigens predominantly within the inner parts of the intima. In <b>A</b> to <b>C</b>, the vessel lumen is to the upper left-hand corner. The demarcation between intima and media is indicated by arrowheads.</p

Lipoprotein staining and effect of GPx-1 deficiency on oxLDL induced foam cell formation.

<p><b>A,</b> Immunohistochemical staining of lipoprotein apo B in parallel with staining of macrophages and SMCs in sequential sections of the aortic arch of both GPx-1^−/−ApoE^−/− (upper panels) and ApoE^−/− (lower panels) mice. The vessel lumen is to the upper left-hand corner. The demarcation between intima and media is indicated by arrowheads. <b>B, C,</b> Effect of GPx-1 deficiency on oxLDL induced foam cell formation. After differentiation for 3 days with 10 ng/ml MCSF, mouse peritoneal macrophages were incubated with 5 and 10 µg/ml oxLDL, respectively, for 24 hours. <b>B,</b> Representative photomicrographs of peritoneal macrophages stained with oil-red O (magnification ×20, inserts ×100). <b>C,</b> Quantitative analysis of cellular cholesterol content in mouse peritoneal macrophages. After incubation with oxLDL, the cells were lysed and homogenized and total cholesterol content was quantified by fluorescence measurements. The results were expressed as total cholesterol per cellular protein. Each value represents the mean ± SD of four separate measurements.</p

Effects of ebselen or oxLDL on MAPK phosphorylation and expression of MAPK in mice lesions.

<p><b>A,</b> After pre-incubation for 3 days with 10 ng/ml MCSF, peritoneal macrophages were incubated for 5 min with 10 µg/ml oxLDL with or without MCSF or MCSF with or without ebselen. Cellular protein was extracted and protein samples (0.4 mg/ml) were analyzed by Western blot with specific antibodies: anti-phosphorylated MEK1/2 or anti-MEK1/2 (<b>A,</b> upper panel<b>,</b> right), anti-phosphorylated ERK1/2 or anti-ERK1/2 (<b>A,</b> middle panel<b>,</b> right) or anti-phosphorylated p90RSK or anti-RSK1/2/3 (<b>A,</b> lower panel, right) antibodies (representative experiments). ß-Actin or Actin were used as control. Quantitative results were calculated by band densitometry with the intensity of phosphorylated MEK1/2, ERK1/2, p90RSK normalized to total MEK1/2, ERK1/2, RSK1/2/3 (<b>A,</b> upper, middle and lower panel, left). Data represent mean ± SD of 5–7 separate experiments. * p<0.05 or ** p<0,01 above the histogram indicate statistically significant differences between the different genotypes and below the histogram compared with cells without treatment of MCSF or oxLDL. <b>B,</b> expression of phosphorylated MEK1/2 and ERK1/2 in parallel with staining of macrophages and SMCs in sequential sections of the aortic arch of both GPx-1^−/−ApoE^−/− (upper panels) and ApoE^−/− (lower panels) mice. There is more pronounced expression of phosphorylated ERK1/2 and MEK1/2 both in macrophages and SMCs of GPx-1^−/−ApoE^−/− compared with ApoE^−/− mice. <b>C</b>, representative double immunohistochemical staining for p-p90RSK (nuclei, brown), macrophages or SMCs (red) in ApoE^−/− mice demonstrating expression of p-p90RSK in macrophages rather than in SMCs (arrowheads). The vessel lumen is to the upper left-hand corner. The demarcation between intima and media is indicated by arrowheads.</p

Effects of MCSF on the phosphorylation of MAPKs.

<p>After pre-incubation for 3 days with 10 ng/ml MCSF, peritoneal macrophages were incubated for 5 and 15 min with 10 ng/ml MCSF. Cellular protein was extracted and protein samples (0.4 mg/ml) were analyzed by Western blot with specific antibodies: anti-phosphorylated MEK1/2 or anti-MEK1/2 (<b>A,</b> right), anti-phosphorylated ERK1/2 or anti-ERK1/2 (<b>B,</b> right), anti-phosphorylated p90RSK or anti-RSK1/2/3 (<b>C</b>, right), anti-phosphorylated p38 MAPK or anti-p38 MAPK (<b>D,</b> right) and anti-phosphorylated SAPK/JNK or anti-SAPK/JNK (<b>E</b>, right) antibodies (representative experiments). ß-Actin or Actin were used as control. Quantitative results were calculated by band densitometry with the intensity of phosphorylated MEK1/2, ERK1/2, p90RSK, p38 MAPK and SAPK/JNK normalized to the total MEK1/2, ERK1/2, RSK1/2/3, p38 MAPK and SAPK/JNK (<b>A–E</b>, left panels). Data represent mean ± SD of 3 separate experiments. *, **indicate statistically significant differences (*p<0.05, **p<0.01) compared with cells without MCSF treatment.</p

Phenotypic analysis of atherosclerotic lesions in SRA-TGF-ß1 ApoE^−/− mice.

<p><b>A</b> Box and whisker diagrams (median, interquartile range, minimum, and maximum; n.s.  =  not significant) of the quantification of macrophages (upper panel), SMCs (middle panel) and collagen (lower panel) in atherosclerotic lesions of SRA-TGF-ß1 ApoE^−/− females and isogenic ApoE^−/− controls after 8, 16 and 24 weeks on the WTD, respectively. <b>B</b> Representative histological slides of atherosclerotic lesions located in the inner aortic arch intima (lesser curvature) of SRA-TGF-ß1 ApoE^−/− mutants and ApoE^−/− controls after 24 weeks on the WTD. The slides have been stained for macrophages, SMCs, and collagen by using a rat anti-mouse F4/80 antibody (upper panel), a mouse anti-smooth muscle α-actin antibody (middle panel), and picrosirius red with subsequent polarization (lower panel). Percent-positive area for macrophages (upper panels, asterisks), SMCs (middle panels, brown-stained areas), and collagen (lower panels, areas with yellow, green, orange, or red polarized colour) were quantified by Photoshop-based image analysis. The aortic lumen is to the upper left corner. The demarcation between intima and media is indicated by black arrowheads. In the lower panel, note that the adventitial tissue (asterisk) also polarizes after picrosirius red staining (internal positive control).</p

Lipoprotein analysis of murine sera.

<p>Group size, body weights, serum cholesterol and serum triglyceride concentrations of CD2-TGFß1 ApoE^−/− and ApoE^−/− mice on ND and WTD. Data are presented as means ± standard deviations.</p><p>*, ** indicate statistically significant differences (* p<0,05, ** p<0,01).</p

Quantification of atherogenesis <i>en face</i> (A) and in the aortic arch (B) in the mouse model of TGF-ß1 overexpressing macrophages after 8 weeks (8 w), 16 weeks (16 w) and 24 weeks (24 w) on the WTD, respectively.

<p><b>A, left panel</b> Box and whisker diagrams (median, interquartile range, minimum, and maximum; n.s.  =  not significant) of aortic plaque area (%) of SRA-TGF-ß1 ApoE^−/− (grey) and ApoE^−/− mice (white). <b>A, right panel</b> Representative Sudan-stained aortas <i>en face</i> of SRA-TGF-ß1 ApoE^−/− and ApoE^−/− mice. <b>B, right panel</b> Hearts of SRA-TGF-ß1 ApoE^−/− and and ApoE^−/− mice were resected, and measurement of plaque size in longitudinal sections of the aortic arch stained with trichrome was performed as follows: a 2-mm segment of the lesser curvature of the aortic arch was defined proximally by a perpendicular axis dropped from the right side of the innominate artery origin (dashed line) and the aortic-arch wall area subtended by this 2-mm stretch of intima (green line) was calculated for each section of all mice by computerized image analysis. In addition, the aortic-arch intima thickness (red line) was determined on this same segment of the lesser curvature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040990#pone.0040990-Blobe1" target="_blank">[1]</a>. IA, innominate artery; LCCA, left common carotid artery; LSA, left subclavian artery. Note, that no statistically significant differences of lesion area could be detected between SRA-TGF-ß1 ApoE^−/− (black circles) and ApoE^−/− (white circles) mice (non-parametric Mann-Whitney U test, n.s.  =  not significant, <b>B, left panel).</b></p

Representative examples of TGFß1 expression in atherosclerotic lesions of SRA-TGF-ß1 ApoE^−/− mice after 24 weeks on the WTD:

<p>Atherosclerotic lesions of the inner aortic arch intima (lesser curvature) were stained with trichrome (upper left panel), rat anti-mouse F4/80 (upper right) for macrophages, mouse anti-smooth muscle α-actin (1A4) (lower left panel) and goat anti-human TGFß1 (lower right panel). The lumen is to the upper left corner. The demarcation between intima and media is indicated by an arrowhead.</p

T-lymphocytes in atherosclerotic lesions.

<p><b>A</b>, Representative immunohistochemical staining of an atherosclerotic lesion located in the inner aortic arch intima (lesser curvature) of a CD2-TGFß1 ApoE^−/− mouse after 24 weeks on WTD. The slide was stained for T-lymphocytes with a monoclonal antibody against CD3 (clone CD3-12, AbD Serotec MorphoSys AbD GmbH, Düsseldorf, Germany) and the number of positively stained cells (asterisks) per mm² was counted (see <b>B</b>). The aortic lumen is to the upper left corner. The demarcation between intima and media is indicated by an arrowhead. <b>B</b>, Box and whiskers diagrams (median, interquartile range, minimum, and maximum) of the quantification of T-lymphocytes in atherosclerotic lesions of CD2-TGFß1 ApoE^−/− females and isogenic ApoE^−/− controls after 24 weeks on WTD (n.s.  =  not significant).</p