Abstract-The transition from stable to rupture-prone and ruptured atherosclerotic plaques involves many processes, including an altered balance between inflammation and fibrosis. An important mediator of both is transforming growth factor (TGF)-␤, and a pivotal role for TGF-␤ in atherogenesis has been postulated. Here, we determine the in vivo effects of TGF-␤ inhibition on plaque progression and phenotype in atherosclerosis. Recombinant soluble TGF-␤ receptor II (TGF␤RII:Fc), which inhibits TGF-␤ signaling, was injected in apolipoprotein E-deficient mice for 12 weeks (50 g, twice a week intraperitoneally) as early treatment (treatment age 5 to 17 weeks) and delayed treatment (age 17 to 29 weeks). In the early treatment group, inhibition of TGF-␤ signaling treatment resulted in a prominent increase in CD3-and CD45-positive cells in atherosclerotic lesions. Most profound effects were found in the delayed treatment group. Plaque area decreased 37.5% after TGF␤RII:Fc treatment. Moreover, plaque morphology changed into an inflammatory phenotype that was low in fibrosis: lipid cores were 64.6% larger, and inflammatory cell content had increased 2.7-fold. The amount of fibrosis decreased 49.6%, and intraplaque hemorrhages and iron and fibrin deposition were observed frequently. TGF␤RII:Fc treatment did not result in systemic effects. These results reveal a pivotal role for TGF-␤ in the maintenance of the balance between inflammation and fibrosis in atherosclerotic plaques.

Objective-Atherosclerosis is a chronic inflammatory disease in which the immune system plays an important role.Neutrophils have not been thoroughly studied in the context of atherogenesis. Here, we investigated neutrophils in the development of murine atherosclerotic lesions. Methods and Results-LDLRϪ/Ϫ mice were given a high-fat diet for different time periods and subsequently atherosclerotic lesions were studied by immunohistochemistry. Staining with anti-Ly-6G monoclonal antibody, a specific marker for neutrophils, revealed a marked accumulation of neutrophils during atherosclerosis development. Neutrophils were observed in the lesion, attached to the cap, and in the arterial adventitia. In addition, at some sites, neutrophil accumulation colocalized with endothelial E-selectin expression. Immunofluorescence double staining with anti-myeloperoxidase and anti-Ly-6G antibodies demonstrated the presence of myeloperoxidase in atherosclerotic lesions and its colocalization with neutrophils. After introducing the high-fat diet, levels of circulating myeloperoxidase in plasma strongly increased, with a peak at 6 weeks and a subsequent decrease to almost normal levels after 16 weeks of diet.Conclusions-We here demonstrate for the first time the presence of neutrophils and myeloperoxidase in murine atherosclerotic lesions. As a major cell type in inflammatory responses the neutrophil may also be an important mediator in the development of atherosclerosis. Key Words:therosclerosis is a multifactorial chronic inflammatory disease with prominent involvement of the immune system. Although incompletely understood, atherogenesis is most likely the result of a complex interplay between various immune and nonimmune cell types. The most abundant immune cells in atherosclerotic lesions are monocyte-derived macrophages and T cells. 1,2 These cells are therefore considered the immunologic key players in atherogenesis and have been extensively studied. In contrast, neutrophils, the principal cellular component of the innate immune system, have received little attention, and their histological presence in atherosclerotic lesions has not been described in detail before.Neutrophils are professional phagocytes, whose main function is to sense and destroy pathogenic organisms. In addition, they are the most prominent leukocytes in acute inflammatory reactions and contribute to host tissue injury in a number of inflammatory conditions, including ischemiareperfusion injury, sepsis, and vasculitis. 3 Importantly, being the key cellular component of the acute inflammatory response, neutrophils are thought to contribute to the initiation and shaping of the immune response. 4 For example, neutrophils generate chemokines that recruit monocytes and dendritic cells and can determine whether macrophages differentiate to a predominantly pro-or antiinflammatory phenotype.Although not extensively investigated, indirect evidence exists implicating a role for neutrophils in the process of atherogenesis. First, atherosclerotic mice deficient in P-sele...

Although it has been demonstrated that carcinogenic environmental polycyclic aromatic hydrocarbons (PAHs) cause progression of atherosclerosis, the underlying mechanism remains unclear. In the present study, we aimed to investigate whether DNA binding events are critically involved in the progression of PAH-mediated atherogenesis. Apolipoprotein E knockout mice were orally (24 wk, once/wk) exposed to 5 mg/kg benzo[a]pyrene (B[a]P), or its nonmutagenic, noncarcinogenic structural isoform benzo[e]pyrene (B[e]P). 32P-postlabeling of lung tissue confirmed the presence of promutagenic PAH-DNA adducts in B[a]P-exposed animals, whereas in B[e]P-exposed and vehicle control animals, these adducts were undetectable. Morphometrical analysis showed that both B[a]P and B[e]P caused an increase in plaque size, whereas location or number of plaques was unaffected. Immunohistochemistry revealed no differences in oxidative DNA damage (8-OHdG) or apoptosis in the plaques. Also plasma lipoprotein levels remained unchanged after PAH-exposure. However, T lymphocytes were increased > or =2-fold in the plaques of B[a]P- and B[e]P-exposed animals. Additionally, B[a]P and to a lesser extent B[e]P exposure resulted in increased TGFbeta protein levels in the plaques, that was mainly localized in the plaque macrophages. In vitro studies using the murine macrophage like RAW264.7 cells showed that inhibition of TGFbeta resulted in decreased tumor necrosis factor (TNF) alpha release, suggesting that enhanced TGFbeta expression in the plaque macrophages contributes to the proinflammatory effects in the vessel wall. In general, this inflammatory reaction in the plaques appeared to be a local response since peripheral blood cell composition (T cells, B cells, granulocytes, and macrophages) was not changed upon PAH exposure. In conclusion, we showed that both B[a]P and B[e]P cause progression of atherosclerosis, irrespective of their DNA binding properties. Moreover, our data revealed a possible novel mechanism of PAH-mediated atherogenesis, which likely involves a TGFbeta-mediated local inflammatory reaction in the vessel wall.

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SummaryIn humans and animal models of atherosclerosis, antibodies against oxidized LDL have been associated with atherosclerotic lesion development. It has been suggested that IgM anti-oxLDL antibodies are anti-atherogenic, whereas IgG anti-oxLDL antibodies are pro-atherogenic. In this study, we examined the relation between IgM and IgG antibody levels and atherosclerosis severity in APOE -/-CD40L -/-mice, which are deficient for IgG and develop moderate advanced atherosclerosis, and compared results with mice developing severe (APOE -/-) or no atherosclerosis (C57Bl/6). Mice were followed in time for anti-oxLDL antibodies while on high-fat diet or normal chow. Anti-oxLDL antibody levels were determined by ELISA. Results revealed that 24-week-old APOE -/-CD40L -/-mice had enhanced IgM anti-oxLDL antibody levels when compared with wild-type mice, but similar levels to those of APOE -/-mice. As expected, IgG anti-oxLDL antibody levels were almost absent in APOE -/-CD40L -/-mice. The transition from early to advanced lesions in APOE -/-mice was reflected by elevated IgM anti-oxLDL antibody levels. IgM anti-oxLDL levels did not further increase during progression to more advanced lesions. No relation was found between IgG anti-oxLDL levels and atherosclerosis severity. In conclusion, the severity of advanced atherosclerosis in mice is not reflected by IgM and/or IgG anti-oxLDL antibody levels. Furthermore, less advanced atherosclerotic lesion development in APOE -/-CD40L -/-mice does not seem to be the result of higher levels of protective IgM anti-oxLDL antibodies. Therefore, our study does not support the idea that the previously observed inconsistency in the relation between anti-oxLDL and atherosclerosis severity is due to differences in antibody isotypes.

ContentsChapter 1 General introduction Chapter 2 IgM anti-oxLDL antibodies, but not IgG anti-oxLDL antibodies, as plasma marker for progressive atherosclerosis Chapter 3 Leukocyte CD40L deficiency affects the CD25 + CD4 T cell population but does not affect atherosclerosis Chapter 4 Transforming growth factor-β mediates balance between inflammation and fibrosis during plaque progression Chapter 5 Depletion of CD4 + CD25 + regulatory T cells enhances the