Involvement of the CD1d-natural killer T cell pathway in neointima formation after vascular injury.

Recent studies have established that the immune system plays an important role in the development of atherosclerosis. However, its role in regulating the arterial response to mechanical injury is less well studied. Arterial injury is associated with local accumulation of antibodies, and mice lacking functional T and B cells exhibit increased neointima formation, indicating that adaptive immune responses to neoantigens in the damaged tissue modulate the vascular repair process. To study the role of lipid antigen presentation in the arterial response to injury, we analyzed neointima formation in mice deficient in the lipid antigen-presenting molecule CD1d using a carotid collar model. As compared with control mice, neointima formation was reduced by >60% (P<0.01) in CD1d-/- mice. Moreover, carotid injury of wild-type C57BL/6 mice was associated with expansion of CD1d-restricted natural killer T cells in the spleen and accumulation of natural killer T cells in the periadventitial space of injured arteries. The results suggest that presentation of lipid antigens through the CD1d-natural killer T cell pathway modulates vascular repair responses.

Inhibition of injury-induced arterial remodelling and carotid atherosclerosis by recombinant human antibodies against aldehyde-modified apoB-100.

OBJECTIVE: The immune system plays an important regulatory role in the development of atherosclerotic plaques and neointima formation following various types of angioplasty. In the present study we investigated the effect of antibodies against aldehyde-modified apolipoprotein B-100 (apoB-100), a component of oxidized LDL, on atherosclerosis and response to arterial injury in mice. METHODS: The ability of a high affinity human recombinant antibody (2D03), specific for malondialdehyde-modified apoB-100, to influence formation of atherosclerosis as well as remodelling and neointima formation after a collar-induced injury of the carotid artery was studied in LDL receptor(-/-) mice over-expressing human apoB-100. RESULTS: The antibody recognized epitopes present in mouse plasma and reduced the plasma level of oxidized LDL by 34%. Antibody treatment inhibited injury-induced restrictive vascular remodelling but did not influence the size of the neointima. Atherosclerosis in the uninjured contra lateral carotid artery was determined by computerized image analysis and the mean plaque area in animals given control IgG1 was 7608+/-10,336 micro m(2). In contrast, essentially no plaques were present in animals treated with the 2D03 antibody (397+/-235 micro m(2), P<0.01 versus control IgG1). CONCLUSIONS: Treatment with antibodies against aldehyde-modified apoB-100 dramatically reduces atherosclerosis and inhibits restrictive vascular remodelling in mice expressing human apoB-100.

Fibulin-2 is present in murine vascular lesions and is important for smooth muscle cell migration.

OBJECTIVE: The vascular extracellular matrix (ECM) can affect smooth muscle cell (SMC) adhesion, migration and proliferation-events that are important during the atherosclerotic process. Fibulin-2 is a member of the ECM protein family of fibulins and has been found to cross-link versican/hyaluronan complexes, an ECM network that has been suggested to be important during tissue repair. In this study we have analysed the presence of fibulin-2 in two different models of murine vascular lesions. We have also examined how the fibulin-2/versican network influences SMC migration. METHODS: Presence of fibulin-2 was analysed by immunohistochemistry in atherosclerotic aortas and in mechanically injured carotid arteries from mice. Fibulin-2 protein levels were also studied by Western blotting during rat aortic SMC phenotypic modulation in vitro. The importance of a fibulin-2/versican interaction for SMC migration was studied in the presence of two inhibiting peptides (FN III 3-5 and aggrecan C-type lectin-like domain). RESULTS: Fibulin-2 is expressed in SMC rich regions of atherosclerotic lesions where it colocalises with versican and hyaluronan. It is also present in injury-induced vascular lesions and is upregulated during SMC phenotypic modulation in cell culture. Moreover, treatments with peptides that block the interaction between versican and fibulin-2 inhibit SMC migration in vitro. CONCLUSIONS: Fibulin-2 can be produced by SMC as a response to injury and may participate in the ECM organisation that regulates SMC migration during vessel wall repair.

B regulatory cells are increased in hypercholesterolaemic mice and protect from lesion development via IL-10.

Whilst innate B1-B cells are atheroprotective, adaptive B2-B cells are considered pro-atherogenic. Different subsets of B regulatory cells (B(reg)) have been described. In experimental arthritis and lupus-like disease, B(reg) are contained within the CD21(hi)CD23(hi)CD24(hi) B cell pool. The existence and role of B(reg) in vascular disease is not known. We sought to investigate the existence, identity and location of B(reg) in vascular disease. The representation of B2-B cell subsets in the spleens and lymph nodes (LNs) of Apolipoprotein E(-/-) (ApoE(-/-)) mice compared to controls was characterised by flow cytometry. Additionally, we utilised a model of neointima formation based on the placement of a perivascular collar around the carotid artery in ApoE(-/-) mice to ascertain whether B cells and B cell subsets confer protection against lesion development. Adoptive transfer of B cells was performed from wild type or genetically modified mice. We showed that CD21(hi)CD23(hi)CD24(hi) B cells are unexpectedly increased in the draining LNs of ApoE(-/-) mice. Adoptive transfer of LN-derived B2-B cells or purified CD21(hi)CD23(hi)CD24(hi) B cells to syngeneic mice reduced lesion size and inflammation without changing serum cholesterol levels. Follicular B2-B cells did not confer protection. IL-10 blockade or transfer of IL10-deficient B cells prevented LN-derived B cell-mediated protection. This is the first identification of a specific LN-derived B2-B(reg) subset that confers IL-10 mediated protection from neointima formation. This may open the way for immune modulatory approaches in cardiovascular disease.

Mobilization of regulatory T cells in response to carotid injury does not influence subsequent neointima formation.

AIM: T cells have been attributed an important role in modulating repair responses following vascular injury. The aim of this study was to investigate the role of different T cell subsets in this context. METHODS AND RESULTS: A non-obstructive collar was introduced to inflict carotid artery injury in mice and subsequent activation of immune cells in draining lymph nodes and spleen were studied by flow cytometry. Carotid artery injury of wild type mice was associated with mobilization of both Th1 type CD4(+)IFNγ(+) and regulatory CD4(+)CD25(+)FoxP3(+) T cells in draining lymph nodes. Studies using FoxP3-green fluorescent protein (GFP) transgenic C57/Bl6 mice demonstrated scattered presence of regulatory T cells in the adventitial tissue of injured arteries as well as a massive emigration of regulatory T cells from the spleen in response to carotid injury. However, deletion of antigen presentation to CD4+ T cells (H2(0) mice), as well as deletion of regulatory T cells (through treatment with blocking anti-CD25 antibodies), did not affect neointima formation. Also deletion of antigen presentation to CD8(+) T cells (Tap1(0) mice) was without effect on carotid collar-induced neointima formation. CONCLUSION: The results demonstrate that carotid artery injury is associated with mobilization of regulatory T cells. Depletion of regulatory T cells does not, however, influence the subsequent repair processes leading to the formation of a neointima. The results also demonstrate that lack of CD8(+) T cells does not influence neointima formation in presence of functional CD4(+) T cells and B cells.

Altered vascular remodeling in osteopontin-deficient atherosclerotic mice.

BACKGROUND: Osteopontin (OPN) is a cell-binding phosphoprotein with proposed functions in atherosclerosis. The aim of this study was to examine how OPN deficiency affects the atherosclerotic process. METHODS: ApoE/LDL receptor/OPN triple knockout (ALO) mice were generated by crossing OPN null mice with ApoE/LDL receptor-deficient (AL) mice. Analysis were made on tissue sections from the aortic arch of 8-, 20- and 34-week female AL and ALO mice and included morphometric measurements, collagen staining, TUNEL staining and immunohistochemistry with antibodies to OPN, macrophages and proliferating cellular nuclear antigen (PCNA). RESULTS: Lesion and media areas were significantly smaller and collagen accumulation in lesions was significantly reduced in 34-week-old ALO mice compared with AL mice. The numbers of proliferating and apoptotic cells were increased in lesions of 34 weeks old ALO mice. Furthermore, the plasma levels of SAA and total cholesterol were significantly decreased in 34 weeks old ALO mice. CONCLUSIONS: The present study shows that OPN deficiency reduces atherogenesis in atherosclerotic mice. The results corroborate and extend recently published findings and also include novel data on the role of OPN in the process of remodeling, inflammation and lipid metabolism.