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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-4530fjev1 20/7/953    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Boesten, L. S. M. Articles by van Vlijmen, B. J. M. Search for Related Content PubMed PubMed Citation Articles by Boesten, L. S. M. Articles by van Vlijmen, B. J. M.

Macrophage retinoblastoma deficiency leads to enhanced atherosclerosis development in ApoE-deficient mice

Lianne S. M. Boesten^*,,1, A. Susanne M. Zadelaar^,, Anita van Nieuwkoop^*,, Lihui Hu^*, Jos Jonkers^||, Bob van de Water^#, Marion J. J. Gijbels^**,, Ingeborg van der Made^**, Menno P. J. de Winther^**, Louis M. Havekes^*,, and Bart J. M. van Vlijmen

^* Department of General Internal Medicine,

Department of Cardiology,

Hemostasis and Thrombosis Research Center, Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands;

TNO-Quality of Life/Gaubius Laboratory, Leiden, The Netherlands;

^|| The Netherlands Cancer Institute, Amsterdam, The Netherlands;

^# Division of Toxicology, Leiden Amsterdam Center for Drug Research; and

^** Department of Molecular Genetics, Cardiovascular Research Institute Maastricht and

Department of Pathology, Maastricht University, Maastricht, The Netherlands

¹Correspondence: Dept. of General Internal Medicine, Leiden University Medical Center, c/o TNO Quality of Life, Gaubius Laboratory, Zernikedreef 9, P.O. Box 2215, Leiden 2301 CE, The Netherlands. E-mail: lsmboesten{at}diac-leiden.nl

SPECIFIC AIMS

The cellular composition of an atherosclerotic lesion is an important determinant of lesion stability. Therefore, modulation of lesion composition is important to reduce the risk of acute coronary syndromes. Cell proliferation and cell death are important processes in regulating the cellular composition of atherosclerotic lesions. The tumor suppressor gene retinoblastoma (Rb) plays a pivotal role in regulating both cell proliferation and cell death in many cell types.

In the current study we aimed to investigate the role of macrophage Rb on the development of atherosclerosis in apolipoprotein E deficient (apoE^–/–) mice. We performed quantitative analysis of aortic atherosclerosis, classification of lesion severity, and detailed immunohistochemical analyses of lesion composition, cell proliferation, and cell death to establish the contribution of macrophage Rb to the development of atherosclerosis.

PRINCIPAL FINDINGS

1. ApoE^–/– mice deficient for macrophage Rb do not show altered levels of blood lipids, blood leukocytes, and inflammatory markers
Conventional Rb knockout mice die during embryogenesis, which hampers studies on the role of Rb in atherosclerosis. Therefore, to investigate the role of macrophage Rb in atherosclerosis development, mice carrying the conditional floxed-Rb gene were combined with LysMCre mice and apoE^–/– mice. This resulted in mice homozygously deficient for Rb in macrophages (LysMCre⁺ Rb^LoxP/LoxPapoE^–/– or Rb^del mice, n=17) and control (LysMCre-negative) littermates that lack apoE (Rb^LoxP/LoxPapoE^–/– or Rb^fl mice n=13). Southern blot analysis revealed that in Cre-recombinase expressing mice the deletion of the floxed allele was almost complete in the heterozygous state (LysMCre⁺Rb^{loxP/wild-type}; Fig. 1 A) and complete in the homozygous state (LysMCre⁺Rb^loxP/loxP; Fig. 1A ) confirming effective deletion of Rb in macrophages.

View larger version (16K): [in this window] [in a new window]   Figure 1. A) Southern blot analysis of thioglycollate-elicited peritoneal macrophages. B) Aortic atherosclerotic lesion area in Rbfl (closed circles, n=13) and Rbdel (open circles, n=17) mice. Line represents mean area for each group. C) Lesion classification of Rbfl (black bars) and Rbdel (white bars) mice. *P < 0.05.

Mean body wt, plasma cholesterol, and triglyceride levels and lipoprotein profiles were not significantly different between Rb^del and Rb^fl mice. Absence of macrophage Rb did not affect hematocrite, leukocyte CD3⁺ (T cell), CD19⁺ (B-cell), and CD11b⁺ (monocyte/granulocyte) concentrations.

2. Macrophage-restricted Rb deletion results in enhanced atherosclerosis development
Male Rb^del and control Rb^fl littermates were fed a cholesterol-rich diet for 12 wk to induce atherosclerosis. Morphometric analysis of aortic root atherosclerotic lesion area revealed a significant 51% increase in Rb^del mice, (P=0.04, Fig. 1B ). In addition, we determined lesion severity. Rb^del mice showed a significant lower incidence of early lesions (P=0.04) and a significant increased incidence of advanced lesions (P=0.04), indicating an enhanced progression of atherosclerosis in Rb^del mice (Fig. 1C ).

In vitro analysis using bone marrow-derived macrophages (BMM) showed that Rb^del and Rb^fl macrophages did not differ in endocytosis of either oxidized LDL, acetylated LDL, or in LPS-induced TNF and Il-10 production. These data show that Rb^del mice have normal modified LDL uptake and are not affected in the production of TNF and Il-10. Plasma levels of the acute-phase inflammatory marker serum amyloid A (SAA) and fibrinogen levels were not significantly affected. These data indicate that macrophage Rb deficiency does not affect the inflammatory status and thereby lesion area in apoE-deficient mice.

In addition, Rb plays a critical role in monocytic differentiation. To evaluate whether the enhanced atherosclerosis in Rb^del mice is due to a defect in monocyte differentiation into macrophages, we immunohistochemically stained spleen sections of Rb^del and Rb^fl mice. No differences were found on the differentiation markers FA-11, Mac1, ERTR9, and F4/80, indicating that macrophage-specific deletion of Rb does not affect the differentiation of monocytes into macrophages.

3. Enhanced atherosclerosis development in Rb^del mice results in advanced lesions poor in macrophages and rich in SMCs
We next investigated the role of macrophage Rb on atherosclerotic lesion composition. Lesion macrophage area in Rb^del mice showed a significant 13% decrease [54.2±12.7 for Rb^del vs. 62.5±13.5 for Rb^fl mice (% of total lesion area) P=0.03]. However, analysis of macrophage area on individual lesions within the same size range (0–50x10³ µm² and 50–150x10³ µm²) showed no difference in macrophage area between Rb^del and Rb^fl mice. These data indicate that the decrease in relative macrophage area in lesions of Rb^del mice is the result of enhanced progression of atherosclerosis, which is a primary characteristic of advanced lesion formation, rather than an additional effect of macrophage-restricted Rb deletion on lesion composition. Analysis of the SMC area showed a significant 46% increase in lesions in the Rb^del mice [4.1±1.8 for Rb^del vs. 2.8±1.6 for Rb^fl mice (% of total lesion area) P=0.05]. Again, analysis of SMC area on individual lesions within the same size range (0–50x10³ µm² and 50–150x10³ µm²) showed no difference between Rb^del and Rb^fl mice, indicating that the increase in SMC area was also a result of the enhanced progression of atherosclerotic lesions.

4. Enhanced atherosclerosis development in Rb^del mice coincides with increased lesional macrophage proliferation
Lesions of Rb^del and Rb^fl mice showed comparable incidence of TUNEL-positive macrophages and SMCs (Fig. 2 A), indicating that macrophage Rb deficiency did not affect lesional apoptosis.

View larger version (39K): [in this window] [in a new window]   Figure 2. A) Apoptosis and (B) proliferation in macrophages (left panel) and SMCs (right panel) in the atherosclerotic lesions of Rbfl (black bars) and Rbdel (white bars) mice. Error bars indicate SEM *P < 0.05. C) Representative Ki-67 stained lesions of Rbfl (left panel) and Rbdel (right panel) mice. Arrows indicate Ki-67 positive cells. Magnification 150x, scale bar 150 µm.

Lesions of Rb^del mice showed a significant 2.6-fold increase in the incidence of Ki-67-positive macrophages (P=0.02, Fig. 2B and C ). The incidence of Ki-67-positive SMC nuclei was not affected by the macrophage Rb genotype (Fig. 2B ). Hence, the increased atherosclerosis in Rb^del mice coincided with increased proliferation of macrophages in the lesions of these mice.

CONCLUSIONS AND SIGNIFICANCE

These studies show that ApoE-deficient mice lacking macrophage Rb displayed accelerated atherosclerosis coinciding with increased macrophage proliferation. The increase in atherosclerosis was characterized by the presence of more advanced lesions that were rich in smooth muscle cells and poor in macrophages. Enhanced atherosclerosis in macrophage Rb deficient mice was independent of modified lipoprotein uptake or cytokine production analyzed in vitro and independent of plasma inflammatory parameters, monocyte differentiation, and lesional apoptosis analyzed in vivo. These studies demonstrate that macrophage Rb is a suppressing factor in the progression of atherosclerosis.

To define the molecular pathways underlying Rb function in vascular disease, our findings may support an initial mechanistic explanation for a role of macrophage Rb in atherosclerosis. We showed that increased macrophage proliferation may underlie the formation of more advanced lesions in Rb^del mice. In general, macrophages produce growth factors, cytokines, chemokines, and metalloproteinases that play an important role in the development and progression of atherosclerotic lesions. This diverse array of these bioactive molecules activates the surrounding endothelium and SMCs. Following, the lesion becomes increasingly complex with the presence of SMCs, lipid-laden macrophages, T-lymphocytes, a necrotic core and cholesterol crystals covered by a fibrous cap. During atherosclerosis development, the macrophage population in Rb^del mice exhibited an increased proliferative state. We hypothesize that this increased proliferative state resulted in enhanced lesion pathology via increased production of growth factors, cytokines, chemokines, and metalloproteinases. This array of proteins stimulated the formation of an advanced atherosclerotic lesion, characterized by a relative decrease in macrophage content and a relative increase in SMC content. Hence, our data suggest that Rb protects against excessive macrophage proliferation and thereby against enhanced atherosclerosis progression.

In conclusion, we demonstrate that deletion of the tumor suppressor gene Rb specifically in macrophages enhances atherosclerosis development. Combined with our previous and comparable findings for p53 in macrophages, we conclude that Rb and p53, in addition to their suppressing function in cancer, have a suppressing function for atherosclerosis development.

View larger version (52K): [in this window] [in a new window]   Figure 3. Schematic drawing illustrating lesion development on macrophage-restricted Rb deletion. Macrophage-restricted Rb deletion leads to enhanced atherosclerosis development characterized by increased lesion area and the presence of more advanced lesions rich in smooth muscle cells and collagen and poor in macrophages

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4530fje

This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-4530fjev1 20/7/953    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Boesten, L. S. M. Articles by van Vlijmen, B. J. M. Search for Related Content PubMed PubMed Citation Articles by Boesten, L. S. M. Articles by van Vlijmen, B. J. M.