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Atherosclerosis is an immune-mediated inflammatory disease of the arterial wall, with both the innate and adaptive immune systems responding to many endogenous and exogenous antigens. Both proatherogenic as well as atheroprotective roles have been identified for the immune system in atherosclerosis. Hence, it is conceivable that an immunomodulatory strategy via active immunization against many of these antigens could potentially alter the natural history of atherosclerosis. This review discusses: 1) the complex role of important components of the innate and adaptive immune systems in atherogenesis; 2) the nature of many antigens that have been tested successfully in vaccine formulations to reduce atherosclerosis in pre-clinical experimental models; and 3) the potential opportunities and challenges for clinical application of vaccination for atherosclerosis in the future.

OBJECTIVES: The present study tested the hypothesis that treatment with human recombinant immunoglobulin G1 (IgG1) antibodies against a specific oxidized low-density lipoprotein (oxLDL) epitope will induce regression of existing atherosclerotic lesions in LDL receptor-deficient mice expressing apolipoprotein B-100 (apoB-100) (Apobec-1(-/-)/LDLR(-/-)). BACKGROUND: Oxidized LDL plays an essential role in the pathogenesis of atherosclerosis. We previously showed that an antibody against oxLDL reduces progression of atherosclerosis in mice. METHODS: Apobec-1(-/-)/LDLR(-/-) mice were fed a high-fat diet until they were 24 weeks and were subsequently transferred to chow. Starting at 25 weeks, mice were given 3 weekly injections of either of 2 recombinant human IgG1 antibodies (IEI-E3 or 2D03) against a malondialdehyde-modified apoB-100 peptide sequence. RESULTS: At 25 weeks, atherosclerotic lesions covered 10.3 +/- 3.7% of the descending aorta. Transfer to chow diet resulted in a modest regression of atherosclerosis over a 5-week period (8.28 +/- 4.36%; p = NS). Antibody treatment induced additional regression of atherosclerosis by 50% (2D03; p = 0.001) and 36% (IEI-E3; p = 0.004) compared with control IgG1. The 2D03 treatment also reduced plaque inflammation, enhanced plaque expression of the adenosine triphosphate-binding cassette transporter A1, and inhibited expression of monocyte chemoattractant protein-1 in cultured monocytes. CONCLUSIONS: Human IgG1 against a specific oxLDL epitope can induce rapid and substantial regression of atherosclerotic lesions, possibly by stimulating lipid efflux and inhibiting macrophage recruitment. These recombinant human antibodies could represent a novel strategy for rapid regression/stabilization of atherosclerotic lesions. (Less)

Objectives. To investigate the mechanisms by which bezafibrate retarded the progression of coronary lesions in the Bezafibrate Coronary Atherosclerosis Intervention Trial (BECAIT), we examined the relationships of on-trial lipoproteins and lipoprotein subfractions to the angiographic outcome measurements. Background. BECAIT, the first double-blind, placebo-controlled, randomized serial angiographic trial of a fibrate compound, showed that progression of focal coronary atherosclerosis in young survivors of myocardial infarction could be retarded by bezafibrate treatment. Methods. A total of 92 dyslipoproteinemic men who had survived a first myocardial infarction before the age of 45 years were randomly assigned to treatment for 5 years with bezafibrate (200 mg three times daily) or placebo; 81 patients underwent baseline and at least one post-treatment coronary angiography. Results. In addition to the decrease in very low density lipoprotein (VLDL) cholesterol (−53%) and triglyceride (−46%) and plasma apolipoprotein (apo) B (−9%) levels, bezafibrate treatment resulted in a significant increase in high density lipoprotein-3 (HDL3) cholesterol (+9%) level and a shift in the low density lipoprotein (LDL) subclass distribution toward larger particle species (peak particle diameter +0.32 nm). The on-trial HDL3cholesterol and plasma apo B concentrations were found to be independent predictors of the changes in mean minimum lumen diameter (r = −0.23, p < 0.05), and percent (%) stenosis (r = 0.30, p < 0.01), respectively. Decreases in small dense LDL and/or VLDL lipid concentrations were unrelated to disease progression. Conclusions. Our results suggest that the effect of bezafibrate on progression of focal coronary atherosclerosis could be at least partly attributed to a rise in HDL3cholesterol and a decrease in the total number of apo B-containing lipoproteins.

Objectives. This study was designed to analyze the functional characteristics of fibroblasts present in aortic valves with degenerative stenosis.Background. Morphologic analysis of degenerative stenosis of tricuspid aortic valves has revealed an extensive interstitial fibrosis.Methods. Stenotic aortic valves collected during aortic valve replacement and control valves collected at autopsy were fixed in formaldehyde, cryosectioned and stained with antibodies against leukocyte markers, HLA-DR and intracellular filaments. Fibroblasts isolated from stenotic valve and skin explants were grown in cell culture, and their proliferative activity was analyzed by cell ocunting and uptake of tritiated thymidine.Results. In the stenotic valves nearly all interstitial cells expressed vimentin, and ~60% of the cells also expressed alpha-actin and desmin. HLA-DR was present on inflammatory cells as well as on one-third of the fibroblast-like cells in the interstitium. Macrophages were found in the interstitium and T lymphocytes close to calcium deposits and in subendothelial areas. In control valves, fibroblasts expressed vimentin but not alpha-actin or desmin. Few inflammatory cells were present in these valves, and HLA-DR expression was restricted to the endothelial surface. In culture, stenotic valve fibroblasts had a reduced ability to proliferate in serum and to activate DNA synthesis in response to growth factors compared with skin fibroblasts from the same patient.Conclusions. The observation that fibroblasts present in aortic valves with degenerative stenosis express smooth muscle cell characteristics and HLA-DR antigen and show signs of cellular senescence in vitro suggests that they are in a state of chronic activation similar to that observed in fibromatosis and scleroderma lesions.

It has been shown that oxidized low density lipoprotein (ox-LDL) stimulates DNA synthesis in smooth muscle cells (SMC). Due to its complex chemical structure it has been difficult to identify the active mitogenic components of ox-LDL. Within the first 48 hours of oxidation we found a 75% reduction of lecithin while lysolecithin was increased 25-fold, lipid peroxide levels increased 8-fold and aldehydes increased 20-fold as determined by the thiobarbituric reactive (TBAR) assay. To identify the components of ox-LDL responsible for stimulation of DNA synthesis, human arterial SMC were exposed to various peroxides, aldehydes, and phospholipids and DNA synthesis was analyzed by autoradiography using radioactive thymidine in corporation in nuclei. Substance % labelled nuclei ± SD p vs Ctl Control (1% serum) 23.3 3.5 LDL 5 μ g/ml 31.0 5.5 NS ox-LDL 5 μ g/ml 75.2 7.8 0.005 lysolecithin 10 μ M 75.3 4.5 0.003 LDL 5 μ g/ml + PhosholipaseA 2 41 U/ml 73.0 3.6 0.0001 H 2 O 2 2OO μ M 39.0 1.7 0.01 The DNA synthesis increased 3 fold with ox-LDL and this effect was replicated by substituting lysolecithin or LDL + Phospholipase A 2 (to generate lysolecithin) for ox-LDL. Peroxides also had a modest stimulatory effect. Malondialdehyde, oxysterols, Iysophosphatidylserine, phosphatidylserine, lecithin, sphingomyelin and sphingosine were without effect. Conclusion The present results indicate that the growth stimulatory effect of oxLDL is due to the formation of lysolecithin and peroxides during the oxidation process.

OBJECTIVESWe investigated whether the effect of bezafibrate on progression of coronary atherosclerosis in the BEzafibrate Coronary Atherosclerosis Intervention Trial (BECAIT) was related to insulin-like growth factor (IGF)-I and glucose-insulin homeostasis.BACKGROUNDBECAIT, the first double-blind, placebo-controlled, randomized, serial angiographic trial of a fibrate compound, demonstrated that progression of focal coronary atherosclerosis in young patients after infarction could be retarded by bezafibrate treatment.METHODSThe treatment effects on serum concentrations of IGF-I and insulin-like growth factor binding protein (IGFBP)-1, as well as on basal and postload glucose and insulin levels, were examined, and on-trial determinations were related to the angiographic outcome measurements.RESULTSBezafibrate treatment resulted in a significant reduction of serum IGF-I levels, both at two and five years, and on-trial serum IGF-I levels were directly related to changes in both minimal lumen diameter (r = 0.25, p < 0.05) and mean segment diameter (r = 0.29, p < 0.05). In contrast, none of the available indexes of insulin resistance (homeostasis model assessment estimate, basal and postload plasma insulin concentrations and serum IGFBP-1 levels) were related to the angiographic changes, nor were they significantly affected by bezafibrate treatment. Multiple stepwise regression analysis showed that the relation between on-trial serum IGF-I level and coronary artery disease (CAD) progression was independent of baseline angiographic score, age, body mass index, serum lipoprotein and plasma fibrinogen concentrations and measures of glucose–insulin homeostasis.CONCLUSIONSIGF-I could be implicated in the progression of premature CAD, and a reduction of serum IGF-I concentration could account partly for the effect of bezafibrate on progression of focal coronary atherosclerosis.

OBJECTIVESTo examine the effect of a polymeric-based periadventitial delivery of a nitric oxide (NO)-releasing diazeniumdiolate, spermine/NO (SPER/NO), on balloon injury-induced neointimal hyperplasia in rat ileofemoral arteries.BACKGROUNDReduced local bioavailability and adverse side effects limit systemic administration of NO to modulate vascular response to injury.METHODSA polylactic-polyglycolic acid polymeric matrix containing 2.5% SPER/NO (w/w) was applied around the injured arteries. Quantitative histomorphometry was performed at day 14, proliferating cell nuclear antigen (PCNA) immunohistochemistry at day 3 to assess effects on smooth muscle proliferation and electrophoretic mobility shift assay to evaluate effects on transcription factor, nuclear factor-kappaB (NF-kappaB).RESULTSTreatment with SPER/NO reduced the intimal area (0.011 ± 0.009 vs. 0.035 ± 0.006 mm2control, p < 0.01) and the intima to media ratio (0.089 ± 0.062 vs. 0.330 ± 0.057 control, p < 0.005). Spermine/nitric oxide produced a profound inhibition of PCNA-positive cells (>75%, p < 0.005) and significantly suppressed the injury-induced activation of NF-kappaB. Vascular cyclic guanosine monophosphate (cGMP) levels were elevated after treatment with the SPER/NO (0.28 ± 0.03 vs. 0.17 ± 0.02 pmol/mg tissue control, p < 0.01). The inhibitory effects on neointimal proliferation were localized to the site of application of SPER/NO and were not associated with any changes in platelet aggregation or bleeding time. Neither SPER nor polymer alone had any significant effects on any of the variables examined.CONCLUSIONSPolymeric-based perivascular delivery of a NO donor produces a marked localized inhibition of neointimal proliferation in balloon-injured arteries. This phenomenon is associated with suppression of NF-kappaB activation and elevation of the vascular cGMP at the site of injury.

Objectives. Cell-specific antibodies were used to identify immunocompetent cells in a comparison of valves from patients who had symptomatic tricuspid aortic stenosis with subjects who had no evidence of valvular heart disease. Background. Nonrheumatic valvular aortic stenosis is the most common valvular heart disease among adults. The biologic processes involved in the development of this disease are poorly understood. Methods. Tricuspid stenotic aortic valves were obtained from 19 patients undergoing surgery for nonrheumatic valvular aortic stenosis, and 10 control valves were collected at autopsy. The valves were fixed in formaldehyde, cryosectioned and stained with antibodies against fibroblasts, endothelial cells, macrophages, T lymphocytes and interlcukin-2 receptors. A subset of valves were also analyzed with antibodies against T-helper cells and cytotoxic T cells. Results. Stenotic valves were characterized by a basal accumulation of calcium deposits and a cell-rich subendothelial thickening. The immunohistologic analysis indicated that the cells in the subendothelial connective tissue were fibroblasts. T lymphocytes appeared to be the most common cell type in the vicinity of the calcium deposits and were also found close to the endothelial lining of the valves. T-helper cells were more frequent than cytotoxic T cells. Expression of interleukin-2 receptors occurred at the same location as T lymphocytes. Control valves lacked subendothelial thickening and contained only few cells reacting with antibodies against lymphocytes and macrophages. Conclusions. The presence of activated T lymphocytes in tricuspid stenotic valves suggests that immunologic mechanisms may be involved in the etiology of nonrheumatic aortic stenosis.

Objectives. In this study we tested the hypothesis that immunization with homologous oxidized low density lipoprotein (oxLDL) would inhibit the neointimal response to balloon injury in hypercholesterolemic rabbits.Background. Immunization with homologous oxLDL has been shown to markedly reduce aortic atherosclerosis in LDL receptor-deficient as well as cholesterol-fed rabbits; however, the effect of this strategy on the balloon injury-induced neointimal lesion is unknown.Methods. New Zealand White rabbits were immunized with 280 μg of homologous native LDL (n = 5), copper-oxidized LDL (n = 5) or phosphate buffer as control (n = 5) and fed a 1% cholesterol diet. Rabbits were reimmunized after 3 weeks, and balloon injury of the right ileofemoral artery was performed 1 week later. Four weeks after balloon injury, rabbits were killed, and the neointimal lesion area was measured by computerized morphometry after perfusion fixation of the arteries. Circulating antibodies against oxLDL were measured by enzyme-linked immunosorbent assay.Results. In comparison with the control animals, those immunized with oxLDL had a 58% reduction in the neointimal area (0.53 ± 0.13 vs. 1.27 ± 0.26 mm2; p = 0.01). The group immunized with native LDL had a 19% reduction in the neointimal area compared with the control group (p = NS). Circulating cholesterol levels and antibody titers against oxLDL were comparable in the three groups. There was a trend toward reduced immunoreactivity for T cells and oxLDL in the neointima of oxLDL-immunized animals.Conclusions. Hypercholesterolemic rabbits immunized with homologous oxLDL have a markedly reduced neointimal area after balloon injury despite severe hypercholesterolemia. Together with previous work, these data suggest that an immunization strategy (vaccination) against atherosclerosis and restenosis warrants further investigation.

Heart failure (HF) is characterized by myocardial dysfunction resulting in increased venous and capillary pressure, and extravascular fluid accumulation when the capacity of the lymphatic system is insufficient. Vascular endothelial growth factor D (VEGF-D) activates VEGF receptors 2 and 3 and is a

Sackgreund: Arterial injury initiates an immune-modulated response but serum levels of interferon-~' (IFN~) after injury has not been characterized. We tested the effect of adoptive transfer of T cells from wild type mice or administration of exogenous IFN-y on the response to injury in B and T cell deficient Rag-1 KO mice. Methods: Carotid artery cuff injury was induced in Rag-1 KO and wild type mice. Serum IFN-~, levels were quantified by ELISA. T cell enriched splenocytes from wild type mice were injected i.v. (2-4 x 107 cells) into Rag-1 KO mice 48 hours prior to injury. Another group of Rag-KO mice was injected with murine IFN-~, (20, 000 units, every other day) starting on the day of injury. Neointimal area was measured 21 days after injury. Results: Basal serum IFN-y in wild type mice was 297.8_+123.4 pg/ml (n--4), and undetectable in 3 of 4 mice tested 24 hours after injury. IFN-~' levels returned to baseline levels 3 days after injury (272_+195.5 pg/ml; n=3). Rag-1 KO mice had low levels of IFN-~' (44.3+41.8 pg/ml; n=3), which was increased after adoptive T cell transfer (464.5_+404.2 pg/ml; n=4) indicating that the T cells were viable. Neointimal area in microns eq