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Background: Garlic (Allium sativum) has been considered to exhibit therapeutic features for many years. The effects of garlic on levels of serum lipids and on atherosclerosis have been investigated extensively. We have previously demonstrated that allicin, an active component of garlic, exerts a beneficial effect on lipid profile in hyperlipidemic rabbits., Objective: To investigate the effects of allicin on formation of fatty streaks (atherosclerosis) and lipid profile in mice., Methods: Allicin was extracted from garlic and kept in a buffer citrate solution at 4 degrees C. Sixty C57BL/6 mice were fed Paigen diet (17% fat, 1.25% cholesterol) for 15 weeks. Thirty randomly selected animals were administered allicin solution (9 mg/kg) and 30 were administered placebo. Blood lipid profile was evaluated five times during the study. At the end of the 15-week period, the animals were killed and the aortic sinus was evaluated for formation of fatty streaks (atherosclerosis)., Results: We observed no statistically significant differences between blood lipid profiles of groups. Microscopic evaluation of aortic sinus formation of fatty streaks (atherosclerosis), however, showed that values for mice in the allicin-treated group were significantly lower: areas of formation of fatty streaks (atherosclerosis) were 13,440 +/- 3310 and 23,410 +/- 3723 micron 2, respectively, for allicin-treated and control mice (means +/- SEM; P = 0.023)., Conclusions: These results indicate that allicin reduces formation of fatty streaks (atherosclerosis) in hyperlipidemic mice. These changes do not seem to occur through an alteration in blood lipid profile.

Background: The assumption that atherosclerosis involves an autoimmune response to oxidized LDL (oxLDL) is based on the presence of immunocompetent cells and immunoglobulin deposition in the atherosclerotic lesions by successful immunomodulation of the atherosclerotic process and by inhibition of experimental atherosclerosis by antioxidants. The Cellscan system is a multiparameter laser-based static cytometer that enables repeated monitoring of the fluorescence intensity (FI) and polarization (FP) of individual living cells. Analysis of intracellular fluorescein fluorescence polarization (IFFP) has previously been used to define activated lymphocyte population., Hypothesis: In this study, the Cellscan apparatus has been used to monitor cellular response to oxLDL in patients with atherosclerosis and in controls., Methods: The FI and FP of fluorescein diacetate (FDA)-labeled peripheral lymphocytes were measured following exposure to oxLDL in vitro. Using cluster analysis we were able to identify subpopulations of cells that were characterized by their FI and FP. Forty-two subjects were studied: 22 patients with severe coronary heart disease and 22 control individuals, either healthy or with other diseases., Results: Fluorescence intensity of fluorescein-labeled peripheral blood lymphocytes (PBL) was markedly decreased upon exposure to high doses (> 25 micrograms/ml) of oxLDL concurrently with an increase in FP. A specific and dose-dependent reduction in FP of the high-intensity cell subpopulations, accompanied by higher FI, was evident in patients with ischemic heart disease upon exposure to low doses of oxLDL (up to 25 micrograms/ml). Maximal depolarization was shown upon triggering with 2 micrograms/ml oxLDL. The polarization ratio (the mean polarization value of the specific cell population with and without activation) obtained for patients' lymphocytes was significantly lower (p < 0.01) than that of the control group (0.936 +/- 0.05 and 1.028 +/- 0.055, respectively)., Conclusion: These data suggest that PBL from patients with active ischemic heart disease show an increased reactivity to oxLDL. A 73% positivity rate was found for ischemic heart disease patients compared with 5% in the control subjects. One of the future prospects of this study might be the advent of a simple and rapid noninvasive test that could assess the extent of atherosclerosis, and possibly even the response to therapy, by monitoring the reactivity of PBL to oxLDL.

Atherosclerosis is a multifactorial process, the hallmark of which is fat deposition in the vessel wall. Autoimmune factors have recently been shown to play an important role in the initiation and progression of atherosclerosis; candidate autoantigens are oxidized lipids and heat shock proteins. beta2-glycoprotein I (beta2-GPI) is a highly glycosylated plasma protein that serves as a major antigenic target for autoimmune type antiphospholipid antibodies. Its major relevant property is binding to negatively charged phospholipids/surfaces. In the set of studies presented in this paper, we provide evidence pointing towards beta2-GPI as an influential determinant in murine and human atherogenesis. Thus, immunization of transgenic atherosclerosis-prone mice (apolipoprotein E and low-density lipoprotein receptor knockouts) with human beta2-GPI results in a brisk and sustained respective response that extends to cross-react with the 'self' murine beta2-GPI. Atherosclerosis is accelerated in both strains concomitant with the infiltration of CD4 lymphocytes in the aortic sinus of the mice. When human plaques were studied, it was found that beta2-GPI resides in the subendothelial regions and co-localizes with CD4 lymphocytes. Thus, the immune response towards beta2-GPI may play an important role in atherogenesis, serving as a possible target for antigen specific therapies., (Copyright 1999 Academic Press.)

Magnesium, an important cofactor of more than 300 enzymes, has previously been found to modulate blood lipid levels, atherogenesis and atherosclerosis in rabbits, when added to their diet. The aim of this study was to examine whether magnesium fortification of drinking water, without a change in diet content, can affect atherogenesis. The study included six groups of LDL-receptor-deficient mice. The mice received either distilled water or water containing 50 g of magnesium sulfate per liter. In the first (12 weeks) and second (6 weeks) stages of the experiment, the mice received low- and high-cholesterol diets, respectively. At the end of each stage, blood was drawn for the determination of plasma magnesium, calcium and lipid levels. In addition, the extent of atherosclerosis was determined at the aortic sinus. In both males and females, magnesium fortification was associated with higher levels of plasma magnesium (50 and 37% increase, respectively), without any differences in plasma calcium content. The extent of atherosclerosis at the aortic sinus in the male mice that received high levels of magnesium was a third of that of the male mice that received distilled water. However, these differences were not found in the female groups. Surprisingly, the female mice that received water fortified with magnesium had higher levels of cholesterol after stage 2, whereas no differences regarding plasma lipid levels were found among the male mice. These results confirm that magnesium fortification of drinking water is capable of inhibiting atherogenesis in male LDL-receptor-deficient mice. The mechanisms of action are yet to be discovered, and are probably not related to diminished lipid excretion, but possibly to the prevention of calcium influx into vascular smooth muscle cells, elevated antioxidative capacity, or other yet undetermined mechanisms., (Copyright 2000 S. Karger AG, Basel.)

Although lipid oxidation plays a major role in atherogenesis, the role of antioxidants in the prevention and treatment of the process is not clear. Apolipoprotein (apo) E-deficient mice develop spontaneous atherosclerotic lesions in major arteries. The presence of oxidized lipoprotein epitopes in the lesion suggests that oxidation reactions are involved in atherogenesis in this mouse model, but the inhibitory effect of antioxidants on atherogenesis in the model is controversial. To test the effect of dietary antioxidants on atherogenesis, male apoE-deficient mice (n=15) were fed a standard chow diet supplemented with 0.05% alpha-tocopherol and 0.05% all-trans beta-carotene. A control group (n=15) received no antioxidant supplement. At the end of the trial, mice consuming vitamins had 5x more plasma vitamin E but undetectable beta-carotene levels. However, liver levels of the beta-carotene metabolite, retinyl palmitate, were higher in antioxidant-treated mice compared with control mice. The antioxidants had no effect on lipoprotein or on plasma anti-oxidatively modified low density lipoproteins (anti-oxLDL) antibody levels. The vitamins had a small but insignificant effect on lipoprotein resistance to ex vivo oxidation, determined by a longer lag period of conjugated diene formation. Atherosclerosis, determined by the lesion size at the aortic sinus, was insignificantly suppressed in antioxidant-treated mice (mean area+/-SE, 20 000+/-7129 versus 13 281+/-5861 micrometer(2); P=0.40). The aortic atherosclerotic lesion area was similar in both experimental groups (2.55+/-0.65% and 2.08+/-0.5% of total aortic area in the control and antioxidant group, respectively; P=0.58). The results of the current study suggest that moderate levels of synthetic antioxidant vitamins have no effect on atherogenesis in apoE-deficient mice.

Recent data suggests that autoimmune factors play an important role in the pathogenesis of atherosclerosis. In this context several autoantigens have been shown to elicit an immune response that results in accelerated atherosclerotic plaque formation. In the present study, we investigated whether elevated titers of anti-oxidized low density lipoprotein (oxLDL), anticardiolipin and antibodies to beta2-glycoprotein I (beta2GPI) can predict subsequent restenosis in patients undergoing percutaneous transluminal coronary angioplasty (PTCA). A total of 74 consecutive patients (52 males, 22 females) with coronary artery disease were enrolled in the study. All patients underwent successful PTCA prior to which blood was drawn for the antibody analysis. The PTCA was followed by a clinical evaluation. Patients with recurrent chest pains underwent a repeated angiography and 34 of the 74 patients (46%) experienced restenosis. Patients positive for the presence of anti-oxLDL antibodies were more likely to develop restenosis within 6 months when compared with patients with no subsequent restenosis (relative-risk of 1.87; P< 0.05). Presence of anti-oxLDL antibodies was associated with hyperlipidemia (r = 0.25; P < 0.05) but not with other risk factors for atherosclerosis. Positivity for anticardiolipin or anti-beta2GPI antibodies which associate with a prothrombotic state, was not effective in predicting lumen narrowing. Thus, the presence of elevated levels of anti-oxLDL antibodies is associated with a higher risk for coronary restenosis following PTCA.

Background: beta2-Glycoprotein I (beta2GPI) is a major antigenic target of antiphospholipid antibodies, which possesses natural anticoagulant properties. The aim of the present study was to determine its presence and localization within human atherosclerotic plaques and to study its association with endothelial cells and monocyte macrophages in vitro., Methods and Results: Human atherosclerotic lesions were obtained after carotid endarterectomies and studied immunohistochemically with anti-beta2GPI as well as antibodies to CD4/CD8, macrophages, and adhesion molecules. In vitro, human umbilical vein endothelial cells (HUVECs) and U937 (myelomonocytic cell line) cells were investigated for their ability to associate with radiolabeled beta2GPI. We found beta2GPI to be abundantly expressed within the subendothelial regions and intimal-medial borders of human atherosclerotic plaques and to colocalize with CD4-positive lymphocytes. This observation was confirmed by Western blot applied on homogenates of atherosclerotic lesions with anti-beta2GPI antibodies. Both HUVECs and U937 cells bound labeled beta2GPI, and the process was inhibited by oxidized LDL and not by native LDL., Conclusions: The abundant presence of human beta2GPI within the lesions, its association with endothelial cells and macrophages, and its colocalization with CD4-positive lymphocytes suggests that it may serve as a target for an immune-mediated reaction that can influence lesion progression.

Recent data suggest that the immune system is involved in atherogenesis. Thus, interest has been raised as to the possible antigens that could serve as the initiators of the immune reaction. In the current work, we studied the effects of immunization with recombinant heat shock protein-65 (HSP-65) and HSP-65-rich Mycobacterium tuberculosis (MT) on early atherogenesis in C57BL/6J mice fed either a normal chow diet or a high-cholesterol diet (HCD). A rapid, cellular immune response to HSP-65 was evident in mice immunized with HSP-65 or with MT but not in the animals immunized with phosphate-buffered saline (PBS) alone. Early atherosclerosis was significantly enhanced in HCD-fed mice immunized with HSP-65 (n=10; mean aortic lesion size, 45 417+/-9258 microm2) or MT (n=15; 66 350+/-6850 microm2) compared with PBS-injected (n=10; 10 028+/-3599 microm2) or nonimmunized (n=10; 9500+/-2120 microm2) mice. No fatty streak lesions were observed in mice fed a chow diet regardless of the immunization protocol applied. Immunohistochemical analysis of atherosclerotic lesions from the HSP-65- and MT-immunized mice revealed infiltration of CD4 lymphocytes compared with the relatively lymphocyte-poor lesions in the PBS-treated or nonimmunized mice. Direct immunofluorescence analysis of lesions from HSP-65- and MT-immunized mice fed an HCD exhibited extensive deposits of immunoglobulins compared with the fatty streaks in the other study groups, consistent with the larger and more advanced lesions found in the former 2 groups. This model, which supports the involvement of HSP-65 in atherogenesis, furnishes a valuable tool to study the role of the immune system in atherogenesis.

We have previously shown that low density lipoprotein receptor-deficient (LDL-RD) mice immunized with beta2-glycoprotein I (beta2GPI; a target of autoimmune anticardiolipin antibodies) developed enhanced early atherosclerosis, when fed a normal chow diet. The current study was undertaken to evaluate the effect of immunization with beta2GPI and the addition of a high fat diet on the progression of atherosclerosis in the apolipoprotein E (ApoE)-deficient mouse. Six-week-old female ApoE-deficient mice (n = 10) were immunized subcutaneously with either human beta2GPI or with ovalbumin, both emulsified in complete Freund's adjuvant and fed a high fat diet for 6 weeks. The beta2GPI-immunized mice were found to develop accelerated atherosclerosis when compared with their ovalbumin-immunized littermates (aortic lesion area of 137,500 +/- 13,801 vs. 72,444 +/- 14,465 microm2, respectively; p = 0.0067). The beta2GPI-immunized mice developed high titers of anti-beta2GPI antibodies, 10 days after the procedure, which were sustained until the sacrifice. LDL extracted from both study groups displayed similar susceptibility to ex vivo oxidation. These results confirm our previous study in which we found increased atherosclerosis in beta2GPI-immunized LDL-RD mice fed a chow diet. In the current study we show that the proatherogenic effect of beta2GPI immunization is maintained despite high cholesterol levels and is not associated with increased susceptibility of LDL to ex vivo oxidation.

In this study we determined the prevalence and clinical associations of immune-complexes-containing beta-2-glycoprotein I (beta2GPI) in randomly selected SLE patients. We studied 38 consecutive SLE patients attending the Rheumatology Unit. Previous arterial or venous-thrombosis were documented by the appropriate diagnostic test. Lipid profile including total cholesterol, LDL, VLDL, HDL and Lp(a) levels were determined from the sera of the fasting patients. Antibodies to cardiolipin, oxidized LDL and beta2GPI were detected employing ELISA. Beta2GPI containing IgG immune-complexes were assayed by using a dot-blot assay. Fourteen SLE patients (36.8%) were found to be positive for the presence of IgG anti-beta2GPI antibodies. Ten of the SLE patients (26.3%) were found to have high levels of beta2GPI containing immune-complexes. There was a positive correlation between beta2GPI-IC levels and the occurrence of thrombocytopenia in the patients (P < 0.05). Furthermore, patients with SLE and venous thrombosis had higher levels of beta2GPI-IC when compared with thrombosis-free patients or with healthy controls (P < 0.05). Patients with higher Lp(a) levels (> 50 mg/dl) possessed higher levels of beta2GPI-IC as compared with patients with lower Lp(a) concentration (< 20 mg/dl) (P < 0.05). These results suggest that IC-containing beta2GPI can help in defining a subpopulation of SLE patients with increased risk of thrombocytopenia and further aid in resolving mechanisms of immune-mediated tissue damage.

Antiphospholipid antibodies (aPL) are found in a variety of autoimmune diseases, and are thought to predispose to arterial and venous thrombosis. These antibodies, when investigated in different assays in vitro, activate endothelial cells and promote uptake of modified LDL to macrophages. These observations suggest that aPL can contribute to atheroma development by targeting some of the sequential steps that constitute early atherogenesis. If substantiated by large-scale clinical trials, the pro-atherogenic properties of aPL may merit screening and intervention programs in selected populations.

Systemic Lupus Erythematosus (SLE) patients experience premature atherosclerosis. A deranged lipid metabolism and use of immunosuppressive medications accounts partially for the accelerated process. The role of autoimmunity in atherosclerosis has recently been highlighted. Autoantigenic determinants thought to play a role in the development of atherosclerosis include: modified lipoproteins, heat shock proteins and beta2-glycoprotein I (a target of 'autoimmune' anticardiolipin antibodies). In this present work we determined autoimmune markers which may be associated with premature atherosclerotic process found in SLE patients. We have found that antibodies to oxLDL were raised in the sera of lupus patients and cross-reacted with cardiolipin and with beta2GPI. OxLDL containing immune-complexes of the IgG and IgM isotypes were both elevated in the SLE patients as compared with healthy controls. Patients with high Lipoprotein (a) concentrations (>30 mg/dl) had higher levels of IgM oxLDL-containing immune-complexes. IgM but not IgG anti-HSP-65 antibodies were elevated in the lupus patients and levels of oxLDL containing immune-complexes correlated positively with the presence of anti-HSP 65 antibodies. Lysophosphatidylcholine (LPC) is a peroxide-derivative formed during LDL oxidation, was shown to evoke a humoral response in healthy subjects. Antibodies to lysophosphatidylcholine of the IgG but not the IgM isotype were reduced in SLE patients compared with controls, suggesting it may be 'consumed' into oxLDL containing immune complexes. Therefore, SLE patients exhibit a humoral autoimmune response towards the antigenic candidates incriminated in the progression of atherosclerosis. These findings may help identify factors that are involved in accelerating atherogenesis in SLE patients.

Patients with chronic renal failure requiring regular hemodialysis are known to be prone to thromboembolic events due to a hypercoagulable state. Vascular access thrombosis (VAT; including thrombosis of the vascular shunt or graft) represents a serious complication and jeopardizes life in these patients. In the current study, conducted on 81 consecutive patients from the Hemodialysis Unit, we have employed ELISA for the estimation of various autoantibody levels (anti-endothelial cell antibodies, anti-cardiolipin, anti-beta 2GPI and anti-modified LDL antibodies) and correlated them with the occurrence of thromboembolic events in general, and VAT in particular. We have found that the levels of antibodies reactive with human umbilical vein endothelial cells (HUVEC) but not with other EC lines (microvascular or EaHy 929) were significantly higher in hemodialysed patients with VAT in comparison with patients with no VAT (p = 0.001). A weaker but yet positive correlation was observed between the levels of anti-HUVEC and anti-cardiolipin antibodies and the occurrence of thromboembolic events including deep vein thrombosis, pulmonary infarction, cerbrovascular events and VAT (both p-values equal 0.02). Anticardiolipin antibodies (aCL) were not cross reactive with beta 2GPI or with HUVEC. Antibodies to modified LDL, although higher in hemodialyzed patients, did not correlate with thromboembolic events. The results of this study suggest that antibodies to HUVEC may prove as a fairly good marker of VAT in hemodialysis. High levels of aCL are weakly associated with thromboembolic events and antibodies to modified LDL do not correlate with a prothrombotic state.

Background: Immunization with beta2-glycoprotein I (beta2GPI), the probable target of autoimmune anticardiolipin antibodies, results in experimental antiphospholipid syndrome in different mouse strains. The present study was undertaken to evaluate the effect of beta2GPI immunization on the progression of atherosclerosis., Methods and Results: In the first experiment, 3 groups of LDL receptor-deficient (LDL-RD) mice (n=15 per group) were immunized with either beta2GPI or ovalbumin or were not immunized and were fed a chow diet for 12 weeks. In a second experiment, 3 groups of LDL-RD mice (n=10 per group) were immunized similarly and fed an atherogenic diet for 6 weeks. All beta2GPI-immunized mice developed high titers of anti-beta2GPI antibodies as well as a specific lymph node proliferation to beta2GPI. The average cholesterol levels did not differ between the mice fed similar diets, regardless of the immunization protocol. Atherosclerosis was enhanced in the beta2GPI-immunized mice (mean aortic lesion, 26 000+/-5700 microm2) in comparison with their ovalbumin-immunized (mean, 3000+/-1099 microm2; P<0.01) and nonimmunized (mean, 2250+/-700 microm2; P<0.01) littermates. The average lesion size in the beta2GPI-immunized mice fed an atherogenic diet (mean, 98 000+/-8305 microm2) was larger than the ovalbumin-immunized mice (mean, 81 250+/-12 933 microm2; P=NS) or the nonimmunized controls (mean, 75 625+/-7281 microm2; P=NS). The atherosclerotic plaques in the beta2GPI-immunized mice appeared to be more mature, and denser infiltration of CD4 lymphocytes was present in the subendothelium of the aortic sinuses from this group of mice., Conclusions: The results of the present study provide the first direct evidence for the proatherogenic effect of ss2GPI immunization and establish a new model for immune-mediated atherosclerosis.

The role of the immune system in modulating atherosclerosis has recently been the subject of intensive research. Several previous authors have put forward a paradigm of the autoimmune process occurring in the vicinity of the plaque. Two recent studies have shown that immunization of rabbits with homologous modified low-density lipoprotein (LDL) led to suppression of atherosclerosis. In the current study we evaluated the effects of homologous malondialdehyde (MDA)-LDL immunizations on atherogenesis in apo-E-deficient mice. Two groups of female chow-diet-fed, apo-E-deficient mice (n = 10) were either immunized with homologous MDA-LDL or with phosphate buffer saline (PBS) at 2-week intervals. The mice were sacrificed 12 weeks following the primary immunization. The MDA-LDL-immunized mice were shown to develop high titers of anti-MDA-LDL antibodies. Atherosclerosis, determined by the lesion size at the aortic sinus, was significantly suppressed in the MDA-LDL-immunized mice as compared with their littermates immunized with PBS (mean area +/- S.D.; 74000 +/- 17300 microm2 versus 158000 +/- 12800 microm2; P < 0.01). No differences were found between the groups with respect to the cellular composition of the atherosclerotic plaques. The results of this study show that immunization with MDA-LDL has a protective effect in apo-E-deficient mice, and further suggests that this mouse model is suitable for studies of immunomodulation.

To determine the effects of vitamin C on cardiovascular risk factors, we studied dietary vitamin C enrichment in 36 healthy male students consuming a diet high in saturated fatty acids. After a 1-mo run-in period during which the subjects consumed approximately 50 mg ascorbic acid/d (low-C diet), half of the subjects were randomly assigned to receive 500 mg ascorbic acid/d for an additional 2 mo (high-C diet). Plasma ascorbic acid increased from 13.5 micromol/L with the low-C diet to 51.7 micromol/L with the high-C diet. Plasma cholesterol increased slightly with the high-C diet, but not above baseline concentrations. This increase was offset by an increase in the lag period of in vitro LDL oxidation, which correlated with plasma ascorbic acid concentrations (r = 0.735, P = 0.0012). Lipoprotein vitamin E concentrations were unchanged with the two diets. There were no effects on concentrations of fibrinogen or factor VII. The fact that ascorbic acid reduced the in vitro susceptibility of lipoproteins to oxidation provides presumptive evidence for an interaction between aqueous and lipophilic antioxidants (vitamins C and E ) in maintaining the integrity of LDL particles.

Atherosclerosis is a multifactorial disease that involves the arterial system. Recent data suggest that immune and autoimmune factors play a dominant role in mediating the progression of atherosclerosis. Among these factors, humoral response to modified forms of LDL and heat-shock proteins has been shown to be influential. The antiphospholipid syndrome (APS) entails clinical manifestations that result from a hypercoagulable state. Antibodies to phospholipids and to beta2-glycoprotein I have been suggested to confer the tendency to thrombosis. In a set of recent studies, we have been able to show that generation of antiphospholipid antibodies in mice is associated with enhanced atherosclerosis. These findings imply that APS and atherosclerosis may share a common etiologic background, which may have direct implications for the management of both conditions.

Atherosclerosis is a process initiated by accumulation of macrophages in distinct areas of endothelial cell damage and uptake of large amounts of lipids. Recently, it has been shown that the immune system plays an active part in the progression of the atherosclerotic plaque although its precise role has not yet been elucidated. Anticardiolipin antibodies (aCL) are generally found in the sera of patients with the antiphospholipid syndrome (APS) and are associated with a prothrombotic state. Several authors have demonstrated that aCL can activate platelets and endothelial cells as well as increase oxidized low density lipoprotein (LDL) uptake by macrophages. In the present study we sought to assess the effect of immunization with aCL (Ab1, leading to the production of mouse aCL-Ab3) on the progression of atherosclerosis. Two groups of 8-weeks old female LDL-receptor knockout mice (n = 13 per group) were immunized with IgG purified from the serum of an APS patient or with normal human IgG, respectively. The aCL immunized mice developed high titres of 'self' aCL (detected using the standard aCL ELISA) as compared with the normal human IgG immunized mice, whereas no differences were noted between both study groups with respect to the serum lipid levels. The extent of fatty streak formation was significantly higher in the aCL immunized mice in comparison with the human IgG injected mice (mean aortic lesion size of 5308 +/- 471 microns2 vs 1027 +/- 184 microns2, respectively, P < 0.01). The immunohistochemical analysis of the atherosclerotic plaques from both mouse groups did not display differences in cellular composition. The results of the study show that mouse aCL induced by immunization with human aCL from an APS patient enhance atherogenesis in LDL-RKO mice and imply that these antibodies may play a role in atherosclerosis development in patients with the APS.

Atherosclerosis is a histopathological process of a multifactorial origin. Whereas the genetic and biochemical causes received considerable attention, the involvement of the immune system has generally been considered negligible. In recent years evidence has been presented to support the dominant role played by the immune system in atherosclerosis. Two major antigenic determinants against which the immune response may be triggered have been suggested, namely the heat shock protein 60/65 and oxidized low density lipoprotein. The current paper reviews the data regarding the involvement of the immune system in atherogenesis with respect to the antigenic candidates mentioned above.

To develop a system for overexpressing genes in the vascular wall, we created transgenic mice using the reporter gene luciferase and the murine preproendothelin-1 promoter. In vitro analysis suggested that the murine 5'-flanking region contained endothelial-specific elements in a 5.9-kb fragment. Five transgenic mice colonies established from independent founders all exhibited the highest level of luciferase activity in the aorta with up to 8,540 light units per microgram of protein. Immunohistochemistry with anti-luciferase antisera revealed high levels of expression in the endothelial cells of both large and small arteries and lower levels of expression in veins and capillaries. Significant expression was also seen in arterial smooth muscle cells and in select epithelial surfaces which is consistent with the known distribution of endothelin-1 in mammals. The further demonstrate the targeting capability of this system, we overexpressed the lipid-peroxidating enzyme, human 15-lipoxygenase, in the vessel wall of transgenic mice. As with luciferase, expression of active enzyme and immunohistochemical localization in vascular cells were documented in transgenic animals. Hence, this new system can be used to direct expression of molecules to the vascular wall for the purpose of examining the biological significance of either overexpression or inhibition of select proteins.

It is well accepted that high levels of low-density lipoprotein (LDL) cholesterol in the plasma are associated with increased risk of atherosclerosis. The cellular and molecular mechanisms linking the two however, have not been fully resolved. One of the processes involved in atherogensis that has been intensively studied in this regard is the oxidation of LDL. Oxidation may convert LDL into an atherogenic form, which incites an inflammatory and proliferative response characteristic of the atherosclerotic lesion. One of the potential mediators in this process is the lipid peroxidating enzyme 15-lipoxygenase, which has been shown to be induced in the atherosclerotic lesion and is capable of oxidizing LDL. In this article, we review the motivation for looking at mechanisms of LDL oxidation and the proposed involvement of 15-lipoxygenase in the pathogenesis of the disease., (Copyright © 1995. Published by Elsevier Inc.)

Seventy-seven hypercholesterolemic patients participated in a 26-week, multicenter, randomized, double-blind, placebo-controlled study that investigated the efficacy and safety of pravastatin therapy. All patients had primary moderate hypercholesterolemia (total cholesterol 200-300 mg/dl, at the end of a 6-week dietary run-in period) and two additional coronary risk factors. Pravastatin, 20-40 mg/day given at bedtime, reduced total cholesterol by 19-22%, LDL-cholesterol by 24-30%, triglycerides by 10-30% and increased HDL-cholesterol by 9-13%. The drug caused mild elevation in alanine aminotransferase and aspartate aminotransferase. Almost all these elevations were within normal limits and no patient was clinically symptomatic. No other significant differences were observed between the pravastatin and the placebo-treated groups with regard to other adverse effects and to patient compliance and withdrawal. It is concluded that pravastatin has a beneficial effect on the lipid profile and that the drug is safe and well tolerated.

Seventeen male Yeshiva students were randomly allocated to a crossover study with two 12-wk dietary periods of monounsaturated fatty acids (MUFAs) vs a carbohydrate (CHO)-rich diet while concentrations of saturated (SFAs) and polyunsaturated (PUFAs) fatty acids were kept similar. Total plasma cholesterol (TC) decreased significantly by approximately 7.7% and low-density-lipoprotein cholesterol (LDL-C) by 14.4% on the MUFA diet, whereas on the CHO diet no significant change in cholesterol concentrations occurred, in contrast to that predicted by the equations of Keys and Hegsted. Concentrations of high-density-lipoprotein cholesterol (HDL-C) did not change significantly on either diet. On the MUFA diet there was a significantly lower proneness to peroxidation of plasma and LDL lipids and less extensive metabolism of conditioned LDL by peritoneal macrophages. We conclude that dietary MUFAs lower TC and LDL-C concentrations, independently of other dietary fatty acids and in addition may reduce the susceptibility of LDL to oxidative stress.

The effect of fish oil ingestion (10 g MaxEPA/day) on the susceptibility of plasma lipoproteins to peroxidation was examined in 20 smokers (study A and B) and 22 nonsmokers (study C). The subjects were examined at the onset of each study (baseline values), divided into control and experimental groups and reexamined 4 weeks later. Smokers were examined 40 h after abstention from smoking (0 time) and 90 min after acute smoking (4-6 cigarettes). The parameters studied were TBARS, which provide an indication of peroxidative injury, and metabolism of conditioned LDL by macrophages as a biological indicator. These parameters were significantly higher (P less than 0.05-0.001) when the 90 min values of smokers were compared to time 0. After 4 weeks of fish oil ingestion, a significant rise above baseline values (33-50%) in plasma and LDL TBARS was found in smokers examined at time 0 and after acute smoking. Peroxidative modification of LDL isolated from smokers fed fish oil resulted in significantly higher TBARS (34-41%) and its metabolism by macrophages was higher (65-139%) compared to baseline values. In nonsmokers, the baseline values of the above parameters were lower than in smokers. Ingestion of fish oil resulted in a significant rise in TBARS in plasma (33%), LDL (137%), conditioned LDL (36-40%) and metabolism of conditioned LDL (70%) by macrophages. In 6 nonsmokers and 4 smokers, 400 mg of vitamin E/day were given with the fish oil. In the nonsmokers, vitamin E counteracted the effect of fish oil more effectively than in the smokers. In the light of the present results, indiscriminate recommendation of fish oil supplementation to the population at large should be cautioned.

The effect of acute smoking on plasma lipoproteins was studied in seventeen smokers. In study 1, 7 subjects were examined prior to and 2 weeks after supplementation with vitamin C. In study 2, the effect of acute smoking was first determined in 10 additional subjects and subsequently they were divided into 3 groups, 3 and 4 subjects were supplemented with vitamin C or E, respectively, for 4 weeks, and 3 remained untreated. Plasma and LDL TBARS were examined at time zero (i.e., 40-48 h after total abstention from smoking) and at 90 min after acute smoking (5-7 cigarettes). In all 17 subjects examined prior to vitamin supplementation, significantly higher TBARS values were found in plasma, native LDL and LDL conditioned with smooth muscle cells (SMC) when the 90 min values were compared to 0 time. The LDL isolated after 90 min and conditioned with SMC was metabolized more extensively by mouse peritoneal macrophages than its zero time counterpart. The differences between the 0 time and 90 min values were not seen after the subjects had been supplemented with vitamin C for 2 or 4 weeks or with vitamin E for 4 weeks. The present results indicate that acute smoking exerts an oxidative stress on plasma lipoproteins and that higher plasma levels of natural antioxidants, such as vitamins C and E have a protective role.

A clinical report of a 67-year-old woman who developed reversible acute renal failure due to focal glomerulonephritis with severe nephrotic syndrome is presented. The patient required four weeks' dialysis treatment but recovered completely. The role of temporary dialysis and diuretics on the clinical course and the reversibility of the process in old age is discussed.

The effect of cigarette smoking on peroxidation of plasma low density lipoprotein (LDL) was studied in 16 smokers aged 23-56 years; 12 nonsmokers of similar age served as controls. The smokers were asked to refrain from smoking 24-40 h prior to testing. Peroxidation was assessed by determination of thiobarbituric acid reactive substances (TBARS) in fresh plasma and LDL and by an increase in 125I-LDL metabolism by peritoneal macrophages. There was no difference in TBARS in freshly prepared plasma or LDL of smokers and nonsmokers. However, LDL of smokers, conditioned by incubation with bovine aortic smooth muscle cells (SMC), had 2-fold or higher TBARS values when compared to SMC conditioned LDL of nonsmokers. 2-4-fold higher TBARS values were seen also in SMC conditioned LDL when the comparison was made between LDL isolated from plasma before smoking of 6-7 cigarettes (time 0), and LDL of the same individual isolated from plasma 90 min thereafter. The metabolism of SMC conditioned 125I-LDL by peritoneal macrophages was examined; LDL isolated from plasma of smokers at time 0 was metabolized twice as avidly as LDL of nonsmokers and a further increase was seen with LDL isolated 90 min after acute smoking. The present results indicate that cigarette smoking renders plasma LDL more susceptible to subsequent peroxidative modification by cellular elements.