Sara Rattik

IL-22 affects smooth muscle cell phenotype and plaque formation in apolipoprotein E knockout mice

OBJECTIVE IL-22 is a recently discovered cytokine that belongs to the family of IL-10 related cytokines. It is produced by activated T-cells and innate lymphoid cells and has been suggested to be involved in tissue repair. As both inflammation and repair play important roles in atherosclerosis we investigated if IL-22 deficiency influences the disease process in Apoe(-/-) mice. METHODS We generated IL-22(-/-)Apoe(-/-) mice and fed them high-fat-diet for 14 weeks to characterize atherosclerosis development. RESULTS IL-22(-/-)Apoe(-/-) mice exhibited reduced plaque size both in the aorta (p = 0.0036) and the aortic root compared (p = 0.0012) with Apoe(-/-) controls. Moreover, plaque collagen was reduced in IL-22(-/-)Apoe(-/-) mice (p = 0.02) and this was associated with an increased expression of smooth muscle cell (SMC)-α-actin (p = 0.04) and caldesmon (p = 0.016) in the underlying media. Carotid arteries from IL-22(-/-)Apoe(-/-) mice displayed increased expression of genes associated with a contractile SMC phenotype e.g. α-actin (p = 0.004) and caldesmon (p = 0.03). Arterial SMCs were shown to express the IL-22 receptor and in vitro exposure to IL-22 resulted in a down-regulation of alpha actin and caldesmon gene expression in these cells. CONCLUSION Our observations demonstrate that IL-22 is involved in plaque formation and suggest that IL-22 released by immune cells is involved in activation of vascular repair by stimulating medial SMC dedifferentiation into a synthetic phenotype. This response contributes to plaque growth by enabling SMC migration into the intima but may also help to stabilize the plaque.

High Plasma Levels of Heparin-Binding Epidermal Growth Factor Are Associated With a More Stable Plaque Phenotype and Reduced Incidence of Coronary Events

Objective—Rupture of atherosclerotic plaques is the major cause of acute coronary events (CEs). Plaque destabilization is the consequence of an imbalance between inflammatory-driven degradation of fibrous tissue and smooth muscle cell–dependent tissue repair. Proinflammatory factors have been documented extensively as biomarkers of cardiovascular risk but factors that contribute to stabilization of atherosclerotic plaques have received less attention. The present study aimed to investigate whether plasma levels of the smooth muscle cell growth factor epidermal growth factor (EGF), heparin-binding-EGF (HB-EGF), and platelet-derived growth factor correlate with plaque phenotype and incidence of CEs. Approach and Results—HB-EGF, EGF and platelet-derived growth factor were measured in plasma from 202 patients undergoing carotid endarterectomy and in 384 incident CE cases and 409 matched controls recruited from the Malmö Diet and Cancer cohort. Significant positive associations were found between the plasma levels of all 3 growth factors and the collagen and elastin contents of the removed plaques. CE cases in the Malmö Diet and Cancer cohort had lower levels of HB-EGF in plasma, whereas no significant differences were found for EGF and platelet-derived growth factor. After adjusting for cardiovascular risk factors in a Cox proportional hazard model, the hazard ratio for the highest HB-EGF tertile was 0.61 (95% confidence interval, 0.47–0.82; P<0.001). Conclusions—The associations between high levels of smooth muscle cell growth factors in plasma and a more fibrous plaque phenotype as well as the association between low levels of HB-EGF and incident CEs point to a potential clinically important role for factors that contribute to plaque stabilization by stimulating smooth muscle cells.

Induction of T helper 2 responses against human Apolipoprotein B100 does not affect atherosclerosis in ApoE−/- mice

AIMS Immune responses against LDL antigens have been found to play an important modulatory role in atherosclerosis. Immunization with homologous oxidized LDL, as well as human apolipoprotein B100 (ApoB)-derived peptides, inhibits atherosclerosis in hypercholesterolaemic animal models of atherosclerosis. However, the role of antigen-specific T helper 2 (Th2) responses in atherosclerosis remains to be fully clarified. METHODS AND RESULTS ApoE(-/-) mice on high-fat diet were immunized with human ApoB using Alum as an adjuvant at 12, 14, and 16 weeks of age. Alum-injected and non-treated mice were used as controls. At 17 weeks of age, a matrigel plug containing ApoB was placed subcutaneously and T-cell infiltration into the plug as well as the development of aortic root atherosclerotic lesions were analysed after an additional 7 days. Immunization with ApoB resulted in four-fold increased accumulation of effector T cells in ApoB-containing matrigel when compared with control groups. The levels of the Th2 cytokines IL-4, IL-5, and IL-10 were also increased in ApoB-containing matrigel plugs. Moreover, the levels of Th2-associated IgG1 against human and also mouse LDL were increased in the plasma of ApoB-immunized mice. In spite of the induction of a Th2 response partially reacting also with the endogenous LDL, there was no difference in atherosclerosis when compared with the Alum group. CONCLUSIONS This study describes a novel model to study antigen-specific T-cell responses in vivo in mouse models of atherosclerosis. The results suggest that activation of Th2 immunity does not mediate the protective effect of immunization with LDL antigens described previously.

Treatment with a GnRH receptor agonist, but not the GnRH receptor antagonist degarelix, induces atherosclerotic plaque instability in ApoE -/- mice

Androgen-deprivation therapy (ADT) for prostate cancer has been associated with increased risk for development of cardiovascular events and recent pooled analyses of randomized intervention trials suggest that this primarily is the case for patients with pre-existing cardiovascular disease treated with gonadotropin-releasing hormone receptor (GnRH-R) agonists. In the present study we investigated the effects of the GnRH-R agonist leuprolide and the GnRH-R antagonist degarelix on established atherosclerotic plaques in ApoE−/− mice. A shear stress modifier was used to produce both advanced and more stable plaques in the carotid artery. After 4 weeks of ADT, increased areas of necrosis was observed in stable plaques from leuprolide-treated mice (median and IQR plaque necrotic area in control, degarelix and leuprolide-treated mice were 0.6% (IQR 0–3.1), 0.2% (IQR 0–4.4) and 11.0% (IQR 1.0-19.8), respectively). There was also evidence of increased inflammation as assessed by macrophage immunohistochemistry in the plaques from leuprolide-treated mice, but we found no evidence of such changes in plaques from control mice or mice treated with degarelix. Necrosis destabilizes plaques and increases the risk for rupture and development of acute cardiovascular events. Destabilization of pre-existing atherosclerotic plaques could explain the increased cardiovascular risk in prostate cancer patients treated with GnRH-R agonists.

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 (H20 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 (Tap10 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.

Decreased levels of stem cell factor in subjects with incident coronary events.

It has been proposed that vascular progenitor cells play an important role in vascular repair, but their possible clinical importance in cardiovascular disease has not been fully characterized. Vascular endothelial growth factor A, placental growth factor and stem cell factor (SCF) are three growth factors that are important in recruiting vascular progenitor cells. In this study, we investigated the association between the plasma levels of these growth factors and incident coronary events (CEs).

B cells treated with CTB-p210 acquire a regulatory phenotype in vitro and reduce atherosclerosis in apolipoprotein E deficient mice

OBJECTIVE Intranasal immunization with a fusion protein of the ApoB100-derived peptide p210 and the cholera toxin B subunit (CTB-p210) has previously been shown to induce mucosal tolerance and reduce atherosclerosis development, but the exact mode of action remains to be elucidated. Recent studies have indicated an important role for B cells in mucosal tolerance, in particular by induction of regulatory B (Bregs) and T cells (Tregs). In this study, we aimed to investigate if transfer of B cells pulsed with CTB-p210 can protect against atherosclerosis. METHOD AND RESULTS First, we studied if CTB-p210 can induce Bregs and Tregs in vitro. After pulsing B cells from Apobtm2Sgyldlr-/- or Apoe-/- mice with CTB-p210 for 1 h and co-culturing them with naïve T cells for 48 h, we observed increased expression of membrane bound TGFβ/latency-associated peptide (mTGFβ/LAP) on B cells and an increased proportion of CD25hiFoxP3+ Tregs. Adoptive transfer of B cells pulsed with CTB-p210 into high-fat diet-fed Apoe-/- mice at 8, 10 and 12 weeks of age, reduced the plaque area in the aorta at 20 weeks of age as compared with control-treated (CTB-pOVA treated B cells or PBS) mice. Moreover, mice receiving p210-CTB treated B cells had increased levels of anti-p210 IgG antibodies. CONCLUSION Our observations suggest that CTB-p210 pulsed B cells acquire a regulatory phenotype and induce Tregs in vitro. Adoptive transfer of CTB-p210, but not control-treated, B cells into Apoe-/- mice decreased atherosclerosis development.

IL‐23R Deficiency Does Not Impact Atherosclerotic Plaque Development in Mice

Background Interleukin‐23 (IL‐23) has been implicated in inflammatory and autoimmune diseases by skewing CD4+ T helper cells towards a pathogenic Th17 phenotype. In this study we investigated the presence of IL‐23 receptor (IL‐23R)‐expressing cells in the atherosclerotic aorta and evaluated the effect of IL‐23R deficiency on atherosclerosis development in mice. Methods and Results We used heterozygous Ldlr −/− Il23r e GFP / WT knock‐in mice to identify IL‐23R‐expressing cells by flow cytometry and homozygous Ldlr −/− Il23r e GFP / eGFP (Ldlr −/− Il23r −/−) mice to investigate the effect of lack of IL‐23R in atherosclerosis. We demonstrate the presence of relatively rare IL‐23R‐expressing cells in lymphoid tissue and aorta (≈0.1–1% IL23R+ cells of all CD45+ leukocytes). After 10 weeks on a high‐fat diet, production of IL‐17, but not interferon‐γ, by CD4+ T cells and other lymphocytes was reduced in Ldlr −/− Il23r −/− compared with Ldlr −/−controls. However, Ldlr −/− and Ldlr −/− Il23r −/− mice had equivalent amounts of aortic sinus and descending aorta lesions. Adoptive transfer of IL‐23R‐deficient CD4+ T cells to lymphopenic Ldlr −/− Rag1 −/− resulted in dramatically reduced IL‐17‐producing T cells but did not reduce atherosclerosis, compared with transfer of IL‐23R‐sufficient CD4+ T cells. Conclusions These data demonstrate that loss of IL‐23R does not affect development of experimental atherosclerosis in LDLr‐deficient mice, despite a role for IL‐23 in differentiation of IL‐17‐producing T cells.

Apolipoprotein B-100 Peptide p210 Inhibits Proliferation of Naïve T Effector Cells and Promotes Induction of Tolerogenic Antigen Presenting Cells and Regulatory T Cells in Vitro

Objectives: Modulation of immune responses against LDL antigens through therapeutic vaccines represents a possible new approach for prevention of cardiovascular disease. The mode of action of these vaccines remains to be fully characterized but the protective effect of immunization with the apolipoprotein B-100 (apoB-100) derived peptide p210 has in several studies been associated with activation of regulatory T cells. The present study used an in vitro model to study the effect of p210 on immune cells. Methods and results: CD11c+ antigen presenting cells, CD25-CD4+ naive T effector cells and CD25+CD4+ T regulatory cells were isolated from mouse spleens using antibody-coated magnetic beads. Pre-incubation of antigen presenting cells with p210 conjugated to cationized bovine serum albumin (p210-cBSA) down-regulated the expression of CD86 and MHC class II molecules, inhibited proliferation of pre-activated naive T effector cells and stimulated conversion of these cells into regulatory T cells. These effects were shown to partly be mediated through a suppression of the release of IL-12 from antigen presenting cells. Conclusions: The present findings demonstrate that p210-cBSA inhibits proliferation of naive T effector cells and promotes their conversion into regulatory T cells and this is suggested to be associated with a reduced activation status of antigen presenting cells. Taken together these findings suggest that immunization with p210-based vaccines have the capability of inducing tolerogenic APCs that in turn generate regulatory T cells suppressing T effector cell functions.