Overexpression of TGF-ß1 in macrophages reduces and stabilizes atherosclerotic plaques in ApoE-deficient mice.

Although macrophages represent the hallmark of both human and murine atherosclerotic lesions and have been shown to express TGF-ß1 (transforming growth factor β1) and its receptors, it has so far not been experimentally addressed whether the pleiotropic cytokine TGF-ß1 may influence atherogenesis by a macrophage specific mechanism. We developed transgenic mice with macrophage specific TGF-ß1 overexpression, crossed the transgenics to the atherosclerotic ApoE (apolipoprotein E) knock-out strain and quantitatively analyzed both atherosclerotic lesion development and composition of the resulting double mutants. Compared with control ApoE(-/-) mice, animals with macrophage specific TGF-ß1 overexpression developed significantly less atherosclerosis after 24 weeks on the WTD (Western type diet) as indicated by aortic plaque area en face (p<0.05). Reduced atherosclerotic lesion development was associated with significantly less macrophages (p<0.05 after both 8 and 24 weeks on the WTD), significantly more smooth muscle cells (SMCs; p<0.01 after 24 weeks on the WTD), significantly more collagen (p<0.01 and p<0.05 after 16 and 24 weeks on the WTD, respectively) without significant differences of inner aortic arch intima thickness or the number of total macrophages in the mice pointing to a plaque stabilizing effect of macrophage-specific TGF-ß1 overexpression. Our data shows that macrophage specific TGF-ß1 overexpression reduces and stabilizes atherosclerotic plaques in ApoE-deficient mice

T cell-specific overexpression of TGFß1 fails to influence atherosclerosis in ApoE-deficient mice.

Clinical data have indicated a negative correlation between plasma TGFß1 concentrations and the extent of atherosclerosis and have thus led to the hypothesis that the pleiotropic cytokine may have anti-atherogenic properties. T-cells are currently discussed to significantly participate in atherogenesis, but the precise role of adaptive immunity in atherogenesis remains to be elucidated. TGFß1 is known to strongly modulate the function of T-cells, however, inhibition of TGFß1 signalling in T-cells of atherosclerosis-prone knock-out mice failed to unequivocally clarify the role of the cytokine for the development of atherosclerosis. In the present study, we thus tried to specify the role of TGFß1 in atherogenesis by using the murine CD2-TGFß1 transgenic strain which represents a well characterized model of T-cell specific TGFß1 overexpression. The CD2-TGFß1 transgenic mice were crossed to ApoE knock-out mice and quantity and quality of atherosclerosis regarding number of macrophages, smooth muscle cells, CD3 positive T-cells and collagen was analyzed in CD2-TGFß1 ApoE double mutants as well as non-transgenic ApoE controls on both normal and atherogenic diet of a duration of 8, 16 or 24 weeks, respectively. In all experimental groups investigated, we failed to detect any influence of TGFß1 overexpression on disease. Total number of CD3-positive T-lymphocytes was not significantly different in atherosclerotic lesions of CD2-TGFß1 ApoE(-/-) females and isogenic ApoE(-/-) controls, even after 24 weeks on the atherogenic diet. The synopsis of these data and our previous study on TGFß1 overexpressing macrophages suggests that potential effects of TGFß1 on atherosclerosis are most probably mediated by macrophages rather than T-cells

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

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

A Generation and expression analysis of transgenic mice with macrophage-specific TGF-ß1 overexpression.

<p>Comparative quantitative RT-PCR analyses was performed with total lung RNA isolated using Tri-Reagent (Sigma-Aldrich, Taufkirchen, Germany) of at least three 8 weeks old transgenic mice of strains SRA-TGF-ß1-B (B), SRA-TGF-ß1-J (J), SRA-TGF-ß1-L (L) and SRA-TGF-ß1-H (H) (left panel). Strain SRA-TGF-ß1-L (L) was used for additional comparative quantitative RT-PCR analysis of total liver RNA (right panel). In both panels, HPRT was used as housekeeping gene for the normalization of the expression data. The relative quantification of the transcripts was done by the 2^(-ΔΔCt) method. <b>B</b> Demonstration of TGF-ß1 expression by immunohistochemistry in both lung (left panel) and liver tissue (right panel) by using a polyclonal goat anti-TGFß1 antibody. <b>C</b> Group size, body weights, serum cholesterol and serum triglyceride concentrations of SRA-TGF-ß1 ApoE^−/− and ApoE^−/− mice. Data are presented as means ± standard deviation.</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

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

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

Quantification of atherosclerosis in the mouse model of TGFß1 overexpressing T cells.

<p><b>A</b>, Box and whisker diagrams (median, interquartile range, minimum, and maximum; n.s.  =  not significant) of the maximal area of the inner aortic arch intima (lesser curvature) of CD2-TGFß1 ApoE^−/− and ApoE^−/− mice on ND (upper panel) and WTD (lower panel). <b>B</b>, Representative Sudan-stained aortas <i>en face</i> of CD2-TGFß1 ApoE^−/− mice and ApoE^−/− controls on WTD.</p

Interferon-γ Induces Chronic Active Myocarditis and Cardiomyopathy in Transgenic Mice

Chronic heart failure is associated with an activation of the immune system characterized among other factors by the cardiac synthesis and serum expression of proinflammatory cytokines. There is unequivocal clinical and experimental evidence that the cytokine tumor necrosis factor-α is involved in the development of chronic heart failure, but a putative cardiotoxic potential of the proinflammatory cytokine interferon (IFN)-γ remains primarily unknown. To investigate this issue we analyzed the cardiac phenotype of SAP-IFN-γ transgenic mice, which constitutively express IFN-γ in their livers and hence exhibit high circulating serum levels of this cytokine. SAP-IFN-γ mice spontaneously developed chronic active myocarditis, characterized by the infiltration of not only CD4+ and CD8+ T cells but also Mac2+ (galectin 3+) macrophages and CD11c+ dendritic cells, eventually culminating in cardiomyopathy. Echocardiographic analyses exhibited a left ventricular dilation and impaired systolic function induced by IFN-γ overexpression. IFN-γ-mediated cardiotoxicity was associated with high-level cardiac transcription of the proinflammatory cytokines tumor necrosis factor-α and interleukin-12 and the macrophage-attracting chemokines MCP1 and MIP1-α. Myotoxic IFN-γ effects could not be detected in smooth or striated muscle tissue, suggesting cardiomyocellular specificity of the toxic IFN-γ effect. The precise mechanism of IFN-γ cardiotoxicity remains to be elucidated