Search

A major drawback of current approaches to antiangiogenic gene therapy is the lack of tissue-specific targeting. The aim of this work was to trigger endothelial cell-specific apoptosis, using adenoviral vector-mediated delivery of a chimeric death receptor derived from the modified endothelium-specific pre-proendothelin-1 (PPE-1) promoter. In the present study, we constructed an adenovirus-based vector that targets tumor angiogenesis. Transcriptional control was achieved by use of a modified endothelium-specific promoter. Expression of a chimeric death receptor, composed of Fas and TNF receptor 1, resulted in specific apoptosis of endothelial cells in vitro and sensitization of cells to the proapoptotic effect of TNF-alpha. The antitumoral activity of the vectors was assayed in two mouse models. In the model of B16 melanoma, a single systemic injection of virus to the tail vein caused growth retardation of tumor and reduction of tumor mass with central tumor necrosis. When the Lewis lung carcinoma lung-metastasis model was applied, i.v. injection of vector resulted in reduction of lung-metastasis mass, via an antiangiogenic mechanism. Moreover, by application of the PPE-1-based transcriptional control, a humoral immune response against the transgene was avoided. Collectively, these data provide evidence that transcriptionally controlled, angiogenesis-targeted gene therapy is feasible.

Background: 15-Lipoxygenase (15-LO) is a nonheme iron-containing enzyme that catalyzes the peroxidation of fatty acids. Herein, we studied the effect of 15-LO overexpression in the vascular endothelium on thymocyte apoptosis by evaluating thymuses from low-density lipoprotein receptor-deficient (LDL-RD) mice and LDL-RD/15-LO mice. Thymuses were evaluated by immunohistochemistry and by TUNEL whereas in vitro studies were carried out by employing freshly isolated thymocytes from the respective mice and evaluation of apoptosis by propidium iodide and annexin V cytometry., Methods and Results: The apoptotic index in LDL-RD/15-LO mice was significantly higher than in the LDL-RD mice. In the thymic medulla the difference was smaller, although still significant. Freshly isolated thymus cells from LDL-RD/15-LO mice exhibited a higher rate of spontaneous cell death than controls. Incubation of thymus cells in the presence of the cell-permeable caspase-3 inhibitor DEVD-CMK resulted in a decrease in the frequency of apoptotic cells in LDL-RD/15-LO thymocytes, whereas no effect was evident in control thymocytes. The antioxidant N-acetylcysteine causes the increase in apoptosis in both groups., Conclusion: LDL-RD/15-LO mice exhibit increased thymocyte apoptosis both in vivo and in vitro. These findings may suggest a role for 15-LO in the natural selection of thymocytes.

Donepezil hydrochloride is a central acetylcholine esterase inhibitor that is widely used in Alzheimer disease (AD). We have recently observed some differences in lipid profile between occasional cases of Donepezil hydrochloride users (DU) and non-users (DNU). This prompted us to study the levels of plasma lipids in these two groups, cross-sectionally. The medical charts of patients with probable AD were screened for current use of Donepezil hydrochloride and lipids profile, along with other clinical and demographic data. A total number of 105 patients were identified and included in the final analysis. Patients were divided into two groups (DU and DNU). Plasma levels of lipids were recorded. Mann-Whitney or t-test for continuous variables and Fisher exact test for categorical variables were used to test for significant differences between the groups. Regression analysis was applied to identify independently the factors associated with lipid levels. Thirty-three patients were DU and 72 DNU. The two groups differed in terms of age, lipid levels and cognitive level. DU had statistically significant higher levels of triglycerides compared with those not using the drug (P=0.036), higher total cholesterol (P<0.001), higher LDL (P=0.001), and higher VLDL (P=0.029) levels. DU showed statistically significant high odds ratios for having higher cholesterol and triglycerides levels. Multivariate regression analysis showed that there are marked differences between DU and DNU with respect to plasma lipid profile, also after correcting for age and sex. The higher plasma levels of cholesterol and triglycerides may reflect an adverse effect of Donepezil hydrochloride. Alternatively, this may indicate that the effect of the medication may involve lipid metabolism, rather than other proposed mechanisms.

Background: Solid evidence suggests that atheroscleosis is associated with immune reactions. Most of the activated T cells in the plaque are T helper 1 subtype (Th1), which secrete interferon-gamma (IFN-gamma), now generally accepted as a proatherogenic cytokine. Interferon-alpha (IFN-alpha) has been found to inhibit the secretion of IL-12 and IFN-gamma and to increase IL-10 production. It may, therefore, be atheroprotective. The aim of the present study was to clarify the effect of IFN-alpha on atherogenesis in a transgenic mouse model of atherosclerosis. Methods: 8-week-old low-density lipoprotein (LDL) receptor-deficient mice were allocated randomly into treatment and control groups (n=13 each). The treatment group received 1000 units of IFN-alpha i.p. every other day for 5 weeks and the control mice received 0.9% NaCl. The mice were fed a Western diet. Results: The IFN-alpha-treated and the control mice showed a similar weight gain (mean 3.9+/-1.0 g vs. 3.4+/-1.8 g, respectively). Treatment with IFN-alpha significantly increased the plasma cholesterol levels in both treated and untreated mice (mean 31.03+/-5.53 mmol/l vs. 24.91+/-6.03 mmol/l, respectively; p<0.022) as well as the plasma triglyceride levels (mean 4.79+/-1.57 mmol/l vs. 3.10+/-1.85 mmol/l, respectively; p<0.033). The IFN-alpha treated mice had a significantly increased atherosclerotic plaque area (mean 61,590+/-22,368 microm(2) vs. 37,272+/-15,469 microm(2), respectively; p<0.008). Conclusion: The putative atheroprotective effect of IFN-alpha by the decrease in IL-10 and IFN-gamma is abolished by hyperlipidemia. Therefore, the net effect of IFN-alpha in this murine model is the exacerbation of atherosclerosis.

Background: Evidence linking cardiovascular risk factors to aortic valve sclerosis (AVS) has led to the assumption that the latter is an atherosclerosis-like process. However, triglyceride (TG)-rich lipoproteins, an important risk factor for atherosclerosis, have been rarely investigated in connection with AVS., Methods: A cross-sectional study of 246 healthy individuals (mean age 59+/-6 years, 77% men) was conducted. Subjects underwent an echocardiographic assessment and extensive blood lipid measurements, including evaluation of TG-related indices, such as serum apolipoprotein (apo) C(II) and C(III) levels, apo C(III) levels in VLDL+LDL particles, and apo C(III) ratio (C(III) level in HDL/C(III) level in VLDL+LDL)., Results: Twenty-three patients (9.3%) were diagnosed as having AVS. On average, these patients were 5 years older and had higher levels of serum cholesterol, LDL-C and LP(a), compared with non-AVS subjects. In addition, the AVS patients exhibited higher concentrations of serum apo C(II), serum apo C(III) and apo C(III) in VLDL+LDL, and a lower apo C(III) ratio. Adjusting for age and gender, a 1 S.D. increment in apo C(III) in VLDL+LDL was associated with odds ratio (OR) of 1.76 (95% CI: 1.17-2.65) for AVS. Further adjustment for atherosclerotic risk factors did not alter the association appreciably (OR=1.65, 95% CI: 1.06-2.58)., Conclusion: TG-rich lipoproteins may be involved in the early development of AVS. Confirmation in prospective studies is required.

Background: Triglycerides (TG) are carried in the circulation by diverse lipoprotein particles, which vary in their lipid and protein content, metabolism, and atherogenicity. Several indices related to apolipoproteins (apo) CII and CIII blood concentration have been proposed to reflect TG metabolism more accurately than the blood level of TG. In the present study we compared the distribution of those indices in coronary heart disease (CHD) patients and controls.., Methods: Ninety consecutively discharged patients with CHD and 209 healthy controls were included in the analysis. Demographic, clinical, and laboratory characteristics were obtained., Results: The CHD patients differed appreciably from controls in several TG-related variables. After adjusting for cardiovascular risk factors, significant associations were found between CHD and the following: TG, VLDL-C, apo CIII, apo CIII in HDL, apo CIII in VLDL + LDL, apo CII- to- TG ratio, and apo CIII ratio (CIII in HDL/CIII in VLDL + LDL). Further adjustment for HDL-C substantially attenuated the above associations, except for those regarding apo CIII in VLDL + LDL (odds ratio (OR): 1.69 per 1 SD increment, 95%CI: 1.03-2.77) and apo CIII ratio (OR: 0.40 per 1 SD increment, 95%CI: 0.15-1.00)., Conclusion: Our results add to the growing evidence which links apo CIII concentration in VLDL + LDL to CHD. Further confirmation in prospective studies would be required before considering this measurement as a screening tool.

Background: Fatty acid bile acid conjugates (FABACs) are novel synthetic molecules that solubilize cholesterol, prevent cholesterol crystal and gallstone formation, and dissolve pre-existing gallstones in mice. They are thus potential agents for gallstone prevention and treatment. The available knowledge concerning their biliary, systemic or possible toxic effects is, however, incomplete., Aim: To obtain information regarding biliary and systemic effects of FABACs., Methods: Hamsters, rats and mice were administered C20-FABAC intragastrically, and serum and bile chemistries, organ histology, animal wellbeing, and survival were monitored., Results: FABAC feeding (150 mg/kg/day) caused no adverse effects in any of the animal species studied. FABAC did not influence biliary cholesterol, phospholipid, or bile-salt concentrations in mice. In hamsters, biliary cholesterol concentration decreased slightly, but effects on phospholipids and bile salts were inconsistent. In some mouse strains, FABAC supplementation increased transaminases slightly. In hamsters and rats, transaminases were mainly unaffected or even decreased. Serum alkaline phosphatase, creatinine, albumin and glucose were generally unaffected by FABAC feeding. No gross or histopathological differences between controls and FABAC-fed animals were noted in any of the organs investigated., Conclusions: C20-FABAC given at a pharmacological dose is safe and devoid of any significant toxic effects in three different animal species.

Omapatrilat inhibits both angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP). ACE inhibitors have been shown to inhibit atherosclerosis in apoE-deficient mice and in several other animal models but failed in low-density lipoprotein (LDL) receptor-deficient mice despite effective inhibition of the renin-angiotensin-aldosterone system. The aim of the present study was to examine the effect of omapatrilat on atherogenesis in diabetic and nondiabetic LDL receptor-deficient mice. LDL receptor-deficient male mice were randomly divided into 4 groups (n = 11 each). Diabetes was induced in 2 groups by low-dose STZ, the other 2 groups served as nondiabetic controls. Omapatrilat (70 mg/kg/day) was administered to one of the diabetic and to one of the nondiabetic groups. The diabetic and the nondiabetic mice were sacrificed after 3 and 5 weeks, respectively. The aortae were examined and the atherosclerotic plaque area was measured. The atherosclerotic plaque area was significantly smaller in the omapatrilat-treated mice, both diabetic and nondiabetic, as compared to nontreated controls. The mean plaque area of omapatrilat-treated nondiabetic mice was 9357 +/- 7293 microm2, versus 71977 +/- 34610 microm2 in the nontreated mice (P = .002). In the diabetic animals, the plaque area was 8887 +/- 5386 microm2 and 23220 +/- 10400 microm2, respectively for treated and nontreated mice (P = .001). Plasma lipids were increased by omapatrilat: Mean plasma cholesterol in treated mice, diabetic and nondiabetic combined, was 39.31 +/- 6.00 mmol/L, versus 33.12 +/- 7.64 mmol/L in the nontreated animals (P = .008). The corresponding combined mean values of triglycerides were 4.83 +/- 1.93 versus 3.00 +/- 1.26 mmol/L (P = .02). Omapatrilat treatment did not affect weight or plasma glucose levels. Treatment with omapatrilat inhibits atherogenesis in diabetic as well as nondiabetic LDL receptor-deficient mice despite an increase in plasma lipids, suggesting a direct effect on the arterial wall., (Copyright 2003 Taylor and Francis)

Background: The administration of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists to low-density lipoprotein (LDL)-receptor-deficient mice resulted in a reduction in the atherosclerotic lesion area in male mice, but not in female mice. The male mice also exhibited reduction in insulin resistance while the female mice did not. To further examine the relationship between PPARgamma agonists, insulin resistance and atherosclerosis, we used the model of accelerated atherosclerosis in male apolipoprotein E (apoE)-deficient mice rendered diabetic by low-dose streptozotocin (STZ)., Methods: Male, apoE-deficient mice (n = 48) were randomly divided into four groups. To induce diabetes, two groups received low-dose STZ and two groups served as controls. After diabetes induction, rosiglitazone (a PPARgamma agonist) was administered by oral gavage to one of the diabetic and one of the non-diabetic groups., Results: Rosiglitazone reduced significantly the atherosclerotic aortic plaque area in both diabetic and non-diabetic apoE-deficient mice: 340 +/- 54 vs. 201 +/- 27 micromol2 (p = 0.001) in diabetic mice; 243 +/- 22 vs. 158 +/- 27 micromol2 (p = 0.001) in non-diabetic mice. Also, rosiglitazone reduced the correlation coefficient between plasma glucose and the degree of atherosclerosis (p < 0.0025) without affecting plasma glucose levels. The rosiglitazone-treated mice, both diabetic and non-diabetic, had higher lipid levels., Conclusions: Rosiglitazone-treated animals showed less atherosclerosis despite higher lipid levels and similar glucose levels. These data suggest a direct anti-atherogenic effect of rosiglitazone on the arterial wall.

Magnesium (Mg) modulates blood lipid levels, atherogenesis, and atherosclerosis in rabbits, when supplemented to diet. We have recently reported that a high concentration (50 g/L) of Mg sulfate fortification of drinking water attenuates atherogenesis in male and female LDL-receptor-deficient mice fed a high-cholesterol diet. The aims of the current study were to examine whether lower concentrations and another Mg salt could also have such an antiatherogenic effect. Thirty male LDL-receptor-deficient mice were divided into three groups (n=10 in each group). The mice received either distilled water or water fortified with 0.83 g or with 8.3 g Mg-chloride per liter. In the first (27 wk) and second (5 wk) stages of the experiment, the mice received normal chow and Western-type diet, respectively. Blood was drawn for determination of plasma Mg, calcium, and lipid levels. The extent of atherosclerotic lesions was determined at the aortic sinus. Magnesium-chloride fortification of drinking water did not result in higher plasma Mg concentrations, whereas a trend toward lower plasma calcium concentrations did not reach statistical significance. Even though plasma lipid levels were similar at the beginning and the end of the study, there were decreased plasma cholesterol and triglyceride levels in the Mg groups after stage I. The atherosclerosis extent at the aortic sinus was significantly decreased in the 8.3-g Mg-chloride/L group (23,437 +/- 10,083 micron2) compared with the control group (65,937 +/- 31,761 microm2). There was also a trend toward lower atherosclerosis extent at the aortic sinus in the 0.83-g Mg-chloride/L group. An additional Mg salt (Mg-chloride) fortification of drinking water is capable of inhibiting atherogenesis in male LDL-receptor-deficient mice. That is done in a lower concentration of Mg than previously reported.

Objective: The goal of this study was to explore the efficacy of oral tolerance with heat shock protein (HSP) 65 in two apparently non-overlapping models of murine atherosclerosis., Background: Atherosclerosis is considered to be a chronic inflammatory process. Autoimmune mechanisms have been shown to influence atherogenesis in experimental animal models. Heat shock protein 65 is a candidate antigen thought to drive a proatherogenic immune-mediated response. Mucosal tolerance is a therapeutic means of accomplishing immune unresponsiveness toward a given antigen by feeding it before active induction of the disorder., Methods: Low-density lipoprotein receptor deficient mice were fed with different doses of HSP65 every other day for 10 days. Feeding with either bovine serum albumin (BSA) or phosphate buffered saline (PBS) served as control. One day after the last feeding, mice were challenged either by immunization with heat killed Mycobacterium tuberculosis or by a high fat diet., Results: Lymphocyte reactivity from mice fed with HSP65 and immunized either against HSP65 or M. tuberculosis was significantly reduced in comparison with BSA-fed mice. Moreover, co-incubation of splenocytes-from mice with tolerance induced with HSP65 but not BSA-with HSP65-reactive lymphocytes resulted in the suppression of HSP65 reactivity by the latter cells. Interleukin-4 production by HSP65-fed and immunized mice was increased upon priming with respective protein. Early atherosclerosis was attenuated in HSP65-fed mice, compared with either BSA- or PBS-fed mice, regardless of the method employed to induce fatty streaks (M. tuberculosis immunization or high-fat diet)., Conclusions: Oral tolerance induced with HSP65 could prove to be a novel means of suppressing atherogenesis.

Background: Apolipoprotein (Apo) C(II) plays an essential role in the metabolism of triglyceride-rich lipoproteins (TRLs) by activating lipoprotein lipase. Several studies have suggested that an abnormal concentration of Apo C(II) may serve as a marker for deficient TRL metabolism, a possible cause of coronary heart disease (CHD). The aim of the present study was to evaluate the association between Apo C(II) and the presence of CHD., Methods: A case-control study comparing 352 CHD patients and 395 controls was performed. Demographic, clinical, and laboratory characteristics were determined., Results: The CHD patients had a higher mean concentration of Apo C(II) than controls (5.3 mg/dl compared with 4.2 mg/dl, P < 0.001). Elevated serum Apo C(II) concentration was associated with CHD presence after adjustment for cardiovascular risk factors. The risk factor-adjusted odds ratio (OR) for CHD was 1.60 (95% CI: 1.31-1.94) per 1 mg/dl increment in Apo C(II), compared with a risk factor-adjusted OR of 1.05 (95% CI: 0.85-1.32) per 40 mg/dl increment in triglyceride concentration., Conclusion: Increased serum concentration of Apo C(II) may represent a more sensitive marker of CHD than high serum concentration of triglycerides. Confirmation in cohort studies in required to establish or refute the role of elevated serum Apo C(II) as a risk factor for CHD.

Background: Hyperlipidemia is a major risk factor for coronary heart disease. Reducing low density lipoprotein-cholesterol can significantly reduce the risk of CHD, but many patients fail to reach the target LDL-C goals due to low doses of statins or low compliance., Objectives: To treat high risk patients with atorvastatin in order to reach LDL-C goals (either primary or secondary prevention) of the Israel Atherosclerosis Society., Methods: In this open-label study of 3,276 patients (1,698 of whom were males, 52%), atorvastatin 10 mg was given as a first dose, with follow-up and adjustment of the dose every 6 weeks. While 1,670 patients did not receive prior hypolipidemic treatment, 1,606 were treated with other statins, fibrates or the combination of both., Results: After 6 weeks of treatment, 70% of the patients who did not receive prior hypolipidemic medications and who needed primary prevention reached target LDL-C levels. Interestingly, a similar number of patients who received prior hypolipidemic treatment (other statins, fibrates or both) and who did not reach the LDL-C treatment goals reached the LDL-C goals for primary prevention with atorvastatin. Only 34% of all patients who needed secondary prevention reached the ISA LDL-C target of 100 mg/dl. Atorvastatin proved to be completely safe, only two patients had creatine kinase elevation above 500 U/L, and another six had mild CK elevation (< 500 U/L). None of the patients had clinical myopathy, and only one had to be withdrawn from the study., Conclusion: Atorvastatin is a safe and effective drug that enables most patients requiring primary prevention to reach LDL-C goal levels, even with a low dose of 10 mg. Patients in need of secondary prevention usually require higher doses of statins.

Background: Platelets have been suggested to play a role in the early development of atherosclerosis. As one test of this hypothesis, we assessed whether patients with Glanzmann thrombasthenia who lack platelet glycoprotein alpha(IIb)beta(3) (GPIIb/IIIa) complexes or both alpha(IIb)beta(3) and the more ubiquitous alpha(v)beta(3) cell membrane complexes are protected from development of atherosclerosis., Methods and Results: Seven patients with Glanzmann thrombasthenia, 45 to 66 years of age, underwent bilateral carotid artery ultrasonography and screening for risk factors of atherosclerosis. Findings consistent with early atherosclerosis evaluated by measurement of intima-media thickness and presence of atherosclerotic plaques were observed in 6 of the 7 patients. Intima-media thickness values higher than the 75th and 90th percentiles of age- and sex-matched white control subjects of the Atherosclerosis Risk in Communities (ARIC) study were observed in 30 and 8 of 56 carotid artery measurements, respectively. Five of the 6 patients with signs consistent with early atherosclerosis lacked both alpha(IIb)beta(3) and alpha(v)beta(3) complexes and 1 only lacked alpha(IIb)beta(3)., Conclusions: Glanzmann thrombasthenia does not protect affected individuals from development of atherosclerosis.

Objective: The aim of the current research was to study whether fatty acid bile acid conjugates (FABACs) have a beneficial effect on atherosclerosis progression and blood lipid levels in mice., Methods: C57BL/6 female mice, fed a high-fat Paigen diet, were administered an oral dose of FABAC or DDH2O daily. Quantification of atherosclerotic fatty-streak lesions at the aortic sinus was performed., Results: The FABAC-treated mice showed a significant reduction in the atherosclerotic lesion areas as compared to the control group (p = 0.019). A significant elevation in total cholesterol levels was observed in both the FABAC and control groups. Higher FABAC levels were measured in the high-density lipoprotein fraction as compared to the very-low-density and low-density lipoprotein fractions., Conclusion: Our findings demonstrate that FABACs, given orally, reduce the development of atherosclerosis in mice fed a high-fat high-cholesterol diet, despite a lack of effect on plasma lipid levels., (Copyright 2003 S. Karger AG, Basel)

Background: Human 15-lipoxygenase (LO) and its murine analogue 12/15-LO are capable of directly oxidizing esterified fatty acids in lipoproteins and phospholipids. Because these oxidized products possess atherogenic properties, it was suggested that LOs may be involved in enhancing atherogenesis. Previous in vivo tests of the role of LOs in atherogenesis animal models, however, have yielded conflicting results., Methods and Results: Aiming to study the role of the 12/15-LO in murine atherogenesis, we crossed LDL-receptor-deficient mice (LDL-R(-/-)) with 12/15-LO-knockout mice and evaluated plaque formation 3 to 18 weeks after initiation of a high-fat diet. Atherosclerotic lesions were considerably reduced in the LDL-R/12/15-LO-double-knockout mice compared with LDL-R(-/-) mice at 3, 9, 12, and 18 weeks, at the aortic root as well as throughout the aorta. The cellular composition of plaques from mice deficient in 12/15-LO did not differ with respect to macrophage and T-lymphocyte content compared with plaques from 12/15-LO littermates., Conclusions: 12/15-LO plays a dominant role in promoting atherogenesis in LDL-R(-/-) mice.

Appreciation of the multifactorial nature of atherosclerosis requires a broad understanding of the mechanisms that underlie its pathogenesis. Autoimmune factors have recently been shown to be associated with the initiation and progression of atherosclerosis. In this context, modified lipoproteins were explored because of their de-novo occurrence within the vessel wall, and heat shock proteins are also being reported by several authors as triggers of autoimmune-like reactions that associate with atherosclerosis. Antiphospholipid antibodies in general and anti-beta2-glycoprotein I (beta2GPI) antibodies in particular have been shown to confer a procoagulant tendency in humans, either in the presence or the absence of the antiphospholipid syndrome. These findings and the ability of antibodies to beta2GPI to activate monocytes and endothelial cells led us to consider whether they are proatherogenic. In a series of studies it was shown that inducing an immune response to beta2GPI in atherosclerosis-prone mice accelerated atherosclerosis. We also demonstrated the abundance of beta2GPI in the atheroma, in conjunction with immunopotent cells. Moreover, when beta2GPI-reactive lymph node and spleen cells were transferred to LDL-receptor-deficient mice they promoted fatty streak formation, proving a direct proatherogenic role for beta2GPI-specific lymphocytes. Perhaps the most important implications of the existence of antigen-specific immune reactions within the atheroma is the ability to exploit them for the purpose of selective immunomodulation. Indeed, we have found that inducing immunological tolerance to beta2GPI by prior oral feeding with the antigen resulted in a significant reduction in the extent of atherosclerotic lesions. Thus, beta2GPI is a candidate player in the atherosclerotic plaque, and can possibly be employed as an immunomodulator of plaque progression.

Magnesium (Mg) fortification of drinking water succeeded in inhibition of atherogenesis development in a transgenic model of atherosclerosis-prone mice fed a high-cholesterol content diet. In order to delineate possible mechanisms of action of the anti-atherogenic effect of Mg, the involvement of LDL oxidation was studied. We determined the susceptibility of LDL to Cu+2 oxidation, anti-oxidized LDL antibody levels, and liver content of retinol and retinyl-palmitate. In order to study another possible mechanism we tested platelets interaction with extracellular matrix in both male and female mice with or without Mg fortification of drinking water. No difference was found in susceptibility of LDL to undergo oxidation. Female mice that received Mg had decreased anti-oxidized LDL antibody levels compared with control female mice, while there was no significant difference among male groups. On the other hand, only in the male group with Mg was a higher content of retinol and retinyl-palmitate found in the livers. Platelets coverage area on extracellular matrix was similar between groups. These results suggest that Mg might affect LDL oxidation, and thus atherogenesis.

Objectives: This study was designed to determine the role of cellular and humoral immune responses to heat shock protein 65 (HSP65) in murine atherosclerosis., Background: Inflammatory processes appear to influence the progression of atherosclerosis. Immunization with HSP65 was previously shown to induce arteriosclerosis in rabbits and to enhance fatty-streak formation in mice. However, it has not been demonstrated directly whether HSP65-reactive antibodies and lymphocytes are separately capable of influencing lesion formation., Methods: Low density lipoprotein-receptor deficient (LDL-RD) mice were immunized with HSP65 or control bovine serum albumin (BSA). Lymph-node cells, splenocytes and immunoglobulin G (IgG) were obtained from the immunized mice and transferred separately to six groups of syngenic LDL-RD mice., Results: Adoptive transfer of HSP65-reactive lymph node cells increased fatty-streak formation in comparison with mice treated with BSA-primed cells. Similarly, transfer of splenocytes reactive with HSP65 led to enhanced fatty-streak generation compared with mice injected with BSA-sensitized splenocytes. Repeated intraperitoneal administration of IgG from serum of HSP65-immunized mice (every 10 days) enhanced fatty-streak formation in mice in comparison with their anti-BSA-IgG injected littermates., Conclusions: Antibodies and lymphocytes reactive to HSP65 promote fatty-streak formation in mice, providing direct evidence for the proatherogenic properties of cellular and humoral immunity to HSP65.

Diabetes mellitus is one of the major risk factors for atherosclerosis. In recent years several murine models have been developed in an attempt to reproduce the accelerated atherosclerosis by combining induced hyperglycemia with hyperlipidemia. In the present study we wished to examine the effect of spontaneous hyperglycemia and hyperlipidemia induced by high fat diet on atherosclerosis development and on markers of the immune system in diabetes prone NOD mice. We tested two high fat dietary regimens (with or without cholate supplementation) in female NOD mice that either developed or did not develop diabetes. Plasma fasting glucose, lipid profile, antibodies to oxidized-LDL and glycated-LDL were assessed. The spleens from both groups were evaluated for their proliferative response. The extent of atherosclerosis was assessed at the aortic sinus. It was found that the two high fat dietary regimens were insufficient to elicit atherosclerosis in the diabetic and non-diabetic NOD mice. The diabetic hyperlipidemic NOD mice displayed an increased cellular immune response to glycated-LDL in comparison with their non-diabetic littermates. The immune response towards copper oxidized LDL was similar in both groups despite an increased susceptibility of LDL extracted from diabetic hyperlipidemic mice to undergo copper induced oxidation. We conclude that the NOD mouse is highly resistant to atherosclerosis even in the presence of hyperglycemia-hyperlipidemia and increased susceptibility to copper induced LDL oxidation.

The application of brief periods of heat stress prior to induction of various forms of tissue injury (ischemia-reperfusion, myocardial infarction, endothelial denudation) has been shown to result in preconditioning and attenuation of subsequent damage. Atherosclerosis represents a state of heightened response to injury at the level of the vessel wall, involving endothelial cells, smooth muscle cells, and macrophages. In the current study, we studied the effects of whole body hyperthermia (WBH) on diet-induced atherosclerosis in a murine model. Low-density lipoprotein receptor deficient mice were either exposed to a 30-min WBH (n = 10) or nontreated (n = 7). Animals were given a high-fat ("Paigen"-type) diet to speed the progression of atherosclerosis immediately following WBH for 6 weeks. Aortic and plaque heat shock protein (HSP) 70, suggested to mediate thermotolerance, was assessed by immunohistochemisry and Western blot at different time points following induction of WBH. Aortic sinus plaque formation was significantly accelerated in WBH-treated mice (275,800 +/- 19,540 microm(2) ) in comparison with their control litters (152,100 +/- 18,200 microm(2); P = 0.0004). Plaque composition was also influenced by WBH as lesions were more mature and had an increased proportion of lipid core/fibrous cap accompanied by increased numbers of apoptotic cells. Total cholesterol and triglyceride levels were not affected significantly by WBH. HSP70 protein expression in the aortas was increased 30 min and 6 and 12 h following WBH induction. Thus, induction of WBH, which affords protection in models of arterial injury, appears to have a proatherogenic role in murine atherosclerosis, despite its upregulatory influence on the expression of HSP70., (Copyright 2001 Academic Press.)

Background: Interindividual variability in responses to warfarin is attributed to dietary vitamin K, drug interactions, age, or genetic polymorphism in the cytochrome P4502C9 enzyme (CYP2C9) (allelic variants 2C9*2 and 2C9*3 ) linked with impaired metabolism of the potent enantiomere S-warfarin., Patients and Methods: We quantified the relative effects of age and of simultaneously determined CYP2C9 genotype, plasma warfarin and vitamin K concentrations, and concurrent medications on warfarin maintenance doses in 156 patients at optimized stable anticoagulation., Results: Allele frequencies for CYP2C9*1, CYP2C9*2, and CYP2C9*3 were 0.84, 0.10, and 0.06. Warfarin doses were 6.5 +/- 3.2, 5.2 +/- 2.4, and 3.3 +/- 2.0 mg/d in the 3 genotype groups (P < .0001). Warfarin doses decreased with age as follows: 7.7 +/- 3.7 versus 4.9 +/- 2.9 mg/d at < 50 years and >66 years (P < .001), mainly as a result of decreased plasma warfarin clearance (2.8 +/- 1.4 mL/min versus 1.9 +/- 0.8 mL/min; P < .001). Vitamin K (1.6 +/- 1.1 ng/mL) did not differ among the age or genotype groups. Patients >or=66 years old with the CYP2C9*3 allele required only 2.2 +/- 1.2 mg/d compared with 7.9 +/- 3.7 mg/d in those

Gene therapy directed specifically to the vascular wall, particularly to angiogenic endothelial cells is a prerequisite in vascular disease treatment. Angiogenesis is a major feature in many pathological conditions including wound healing, solid tumors, developing metastases, ischemic heart diseases and diabetic retinopathy. In the present study we developed a tissue-specific gene therapy to the angiogenic blood vessels of tumor metastasis using an adeno-based vector containing the murine preproendothelin-1 (PPE-1) promoter. Genes activated by the PPE-1 promoter were highly expressed in bovine aortic endothelial cells in vitro. Systemic injection of the adenoviral vectors AdPPE-1-luciferase and AdCMV-luciferase to normal C57BL/6 mice, resulted in higher activity of PPE-1 promoter compared with CMV promoter in the aorta and vascularized tissues such as heart, kidney, lung and pancreas. Systemic administration of the adenoviral vector, in mice bearing Lewis lung carcinoma, resulted in high and specific activity of PPE-1 in the new vasculature of primary tumors and lung metastasis. Cellular distribution of the delivered gene revealed highest expression of GFP in angiogenic endothelial cells of the metastasis. We expect that this approach of 'vascular-directed' gene therapy will be applicable to both vascular diseases and cancer.

Background: Case-control and prospective studies indicate that an elevated plasma homocysteine level is a powerful risk factor for atherosclerotic vascular diseases. Certain medications can induce hyperhomocystinemia, such as methotrexate, trimethoprim and anti-epileptic drugs. There are few reports indicating an interaction between lipid-lowering drugs (cholestyramine and niacin) and homocysteine. Recently, an interaction was shown between fenofibrate and benzafibrates (a fibric acid derivative) and homocysteine plasma levels., Objectives: To evaluate the effects of different fibrates on plasma homocysteine levels and to measure the reversibility of this effect., Methods and Results: We investigated the effects of ciprofibrate and bezafibrate on homocysteine levels in patients with type IV hyperlipidemia and/or low high density lipoprotein levels. While a 57% increase in homocysteine was detected in the ciprofibrate-treated group (n = 26), a 17% reduction in homocysteine was detected in the group treated with bezafibrate (n = 12). The increase in homocysteine in the ciprofibrate-treated group was sustained for the 12 weeks of treatment and was partially reversible after 6 weeks of discontinuing the ciprofibrate therapy., Conclusions: These results indicate that an increase in plasma homocysteine levels following administration of fibrates is not a class effect, at least in its magnitude. Moreover, it is reversible upon discontinuation of the treatment.

Background: The significance of antioxidized low-density lipoprotein (oxLDL) antibodies in atherogenesis is not yet clear, and there are conflicting data regarding anti-oxLDL levels in early hypertension., Methods: The levels of anti-oxLDL antibodies were studied in coronary artery disease patients with (n = 82) or without (n = 36) hypertension, in association to other risk factors for coronary artery disease., Results: The levels of anti-oxLDL antibodies did not differ significantly between coronary artery disease patients with or without hypertension. (0.132 +/- 0.146 v 0.153 +/- 0.158 optical density at 405 nm, respectively; P = .48). No significant differences in anti-oxLDL antibodies were found between men and women with and without hypertension, between hypertensive patients with normal and abnormal blood pressure measurements, and between medicated and nonmedicated hypertensive patients. The presence of diabetes mellitus, smoking, and hypercholesterolemia, either solely or in combination, did not result in significant differences in antibody levels in the hypertensive or normotensive patients., Conclusions: Although the levels of oxLDL antibodies might be modified in early hypertension, once advanced coronary artery disease has developed the presence of hypertension does not affect anti-oxLDL levels.

Background: Autoimmune factors have been shown to play a role in atherosclerosis. The aim of this study is to correlate 5 autoantibodies (anticardiolipin, anti-CL, beta2-glycoprotein-I, beta2GPI, phosphatidylcholine, oxidized low-density lipoprotein, oxLDL, endothelial cell) with the presence of coronary heart disease, angiographic findings, and with coronary artery calcification., Methods: The levels of the 5 autoantibodies and a control antifibroblast line of 126 coronary heart disease patients and 20 healthy controls were measured. Fifty-one patients underwent coronary angiography, and 98 patients had coronary artery calcium determination using spiral computerized tomography (dual mode)., Results: Levels of 3 autoantibodies (anti-CL, beta2GPI, oxLDL) were significantly elevated in coronary heart disease patients compared with controls (p < 0.001, p = 0.001, p < 0.001, respectively). Within the subgroup of patients with significant coronary artery stenosis, anti-CL antibodies were also elevated (p = 0.008). No correlation was found between anti-CL, and anti-beta2GPI autoantibody levels and coronary calcium scores as measured by spiral computerized tomography. However, anti-oxLDL antibodies were raised in patients with no calcification detected by spiral computerized tomography, compared with the patients with any coronary calcification (p = 0.046)., Conclusion: Anti-CL, beta2GPI and oxLDL antibodies are elevated in coronary heart disease patients regardless of coronary calcification., (Copyright 2001 S. Karger AG, Basel.)

Objective: The aim of the present study was to compare the accumulation of (125)I-labeled low-density lipoproteins (LDL), oxidized LDL (oxLDL), HDL and BSA in advanced atherosclerotic lesions of apoE-deficient mice., Methods: (125)I-lipoproteins or (125)I-BSA were injected into the tail vein of apoE-deficient mice. Blood clearance of (125)I-lipoproteins and (125)I-BSA and their accumulation in atherosclerotic lesions were assayed., Results: Blood clearance of (125)I-LDL and (125)I-HDL was moderate, and approximately 30% of the injected lipoproteins were present in plasma 24 h following injection. oxLDL was removed much faster from plasma, and less than 10% of (125)I-oxLDL was present in the circulation 30 min after (125)I-oxLDL injection. The clearance of (125)I-BSA from the circulation was slower than the lipoprotein clearance. The highest accumulation of LDL, oxLDL, HDL and BSA was detected in atherosclerotic lesions in the aortic arch and abdominal aorta, while lower accumulation was detected in the less atherosclerotic descending thoracic aorta., Conclusion: Our findings demonstrate that both (125)I-HDL and (125)I-BSA as well as (125)I-LDL are accumulated in atherosclerotic plaques and that they can be used for the detection of atherosclerotic lesions., (Copyright 2002 S. Karger AG, Basel)

Novel risk factors for the progression of atherosclerosis such as C-reactive protein (CRP) and adhesion molecules have stimulated much recent interest in the role of inflammation in atherosclerotic disease. There is also evidence emerging that autoimmunity may have a role in the pathogenesis of atherosclerosis. In this article we explore the evidence for the role of autoimmunity in human atherosclerosis, both in the general population and in the context of the antiphospholipid syndrome. In particular we will focus on several autoantigens, review the evidence for their role in the process of atherosclerosis and the nature of the immune responses.

15-Lipoxygenase (15-LOX) is a lipid-oxidizing enzyme that is involved in cell cycle regulation. To evaluate the effect of 15-LOX on reproduction, we studied transgenic mice that overexpress 15-LOX. The transgene was introduced over the genetic background of the low density lipoprotein receptor deficient mice (LDL-R(-/-)) and reproduction was compared to LDL-R(-/-) mice. We found a lower pregnancy rate in the 15-LOX/LDL-R(-/-) mice as compared to the LDL-R(-/-) mice (62.74 vs. 79.1%, p < 0.01). Additionally, a remarkably higher number of resorptions per pregnancy was found in the 15-LOX/LDL-R(-/-) mice (16.7 vs. 3.27%, p < 0.001) and it was accompanied by a significantly higher activity of the 15-LOX enzyme in these resorptions as compared to other tissues. These findings may implicate a role for 15-LOX in the development of spontaneous abortions., (Copyright 2001 S. Karger AG, Basel)

The very low density lipoprotein receptor (VLDLR) has been proposed to play a role in the delivery of fatty acids to peripheral tissues. However, despite reduced adipose tissue mass in VLDLR-deficient (VLDLR(-)(/-)) mice, this has been difficult to substantiate. In the present study, VLDLR-deficient and VLDLR-overexpressing (PVL) mice were cross-bred onto a low density lipoprotein receptor knockout (LDLR(-)(/-)) background to study the VLDLR under conditions of relatively high serum VLDL and triglyceride levels. Absence of the VLDLR resulted in a significant increase in serum triglyceride levels (1.9-fold) when mice were fed a high fat diet. In contrast, overexpression of the VLDLR resulted in a significant decrease in serum triglyceride levels (2.0-fold) under similar conditions. When kept on a chow diet, a period of prolonged fasting revealed a significant increase in serum triglyceride levels in VLDLR(-)(/-); LDLR(-)(/-) mice (2.3-fold) as compared with LDLR(-)(/-) controls. This could not be attributed to altered apolipoprotein B and VLDL triglyceride production rates. Furthermore, no major differences in nascent VLDL triglyceride content were found between VLDLR(-)(/-); LDLR(-)(/-) mice and LDLR(-)(/-) controls. However, the triglyceride content of circulating VLDL of VLDLR(-)(/-); LDLR(-)(/-) mice (63%) was relatively high as compared with LDLR(-)(/-) controls (49%). These observations suggest that the VLDLR affects peripheral uptake of VLDL triglycerides. In conclusion, under conditions of LDLR deficiency in combination with high fat feeding or prolonged fasting, the effect of the VLDLR on VLDL triglyceride metabolism was revealed.

Abundant data is present to implicate oxidatively modified low-density lipoprotein (oxLDL) in enhanced atherogenesis. Among the factors involved in LDL oxidation, an important role has been attributed to human 15-lipoxygenase (LO) and its murine analog 12-LO. The expression of these peroxidizing enzymes is under the control of cytokines, the principal of which is IL-4. In the present study we tested the hypothesis that knocking out the IL-4 gene from C57BL/6 mice would result in suppression of fatty streaks. For this purpose, we have fed 45 female IL-4 transgenic knockout (IL-4T KO) and 45 wild-type (WT) mice an atherogenic diet for 15 weeks. Consecutive determinations of the lipid profile from both study groups were performed at monthly intervals, and fatty streak formation was assessed at the aortic sinus level, upon sacrifice. The two study groups did not differ significantly with respect to the lipid profile or the uptake and degradation of iodinated oxLDL by their peritoneal macrophages. We found that the endogenous deficiency of IL-4 did not confer protection from early atherosclerosis in the IL-4T KO as compared to their WT littermates (determined at the aortic sinus). Immunohistochemical studies, Western blots and 12/15-LO activity assays revealed the presence and activity of 12/15-LO in macrophages of WT mice as well as in IL-4T KO mice. Both did not differ significantly between the study groups. The data from this study imply that deficiency in IL-4 does not affect early atherosclerosis in C57BL/6 mice fed a high-cholesterol diet.

Atherosclerosis is a multifactorial pathological process. In recent years, the immune factors associated with its initiation and progression have been investigated intensively. Several autoantigens and their respective autoantibodies have been suggested as factors in atherogenesis. This manuscript provides a review of autoantibodies directed towards oxidized low-density lipoprotein (oxLDL), cardiolipin, beta2-glycoprotein-I and heat-shock protein 60/65, and their association with human and murine atherosclerosis.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the key regulatory enzyme in the mevalonate (MVA) pathway, is rapidly degraded in mammalian cells supplemented with sterols or MVA. This accelerated turnover was blocked by N-acetyl-leucyl-leucyl-norleucinal (ALLN), MG-132, and lactacystin, and to a lesser extent by N-acetyl-leucyl-leucyl-methional (ALLM), indicating the involvement of the 26 S proteasome. Proteasome inhibition led to enhanced accumulation of high molecular weight polyubiquitin conjugates of HMGR and of HMGal, a chimera between the membrane domain of HMGR and beta-galactosidase. Importantly, increased amounts of polyubiquitinated HMGR and HMGal were observed upon treating cells with sterols or MVA. Cycloheximide inhibited the sterol-stimulated degradation of HMGR concomitantly with a marked reduction in polyubiquitination of the enzyme. Inhibition of squalene synthase with zaragozic acid blocked the MVA- but not sterol-stimulated ubiquitination and degradation of HMGR. Thus, similar to yeast, the ubiquitin-proteasome pathway is involved in the metabolically regulated turnover of mammalian HMGR. Yet, the data indicate divergence between yeast and mammals and suggest distinct roles for sterol and nonsterol metabolic signals in the regulated ubiquitination and degradation of mammalian HMGR.

Background: It has been proposed that autoimmune factors can influence the progression of atherosclerosis. We have previously shown that immunization of LDL receptor-deficient (LDL-RD mice) with beta(2)-glycoprotein I (beta2GPI; a principal target of "autoimmune" antiphospholipid antibodies) enhances early atherosclerosis. In the present study, we tested the hypothesis that adoptive transfer of beta2GPI-reactive T cells can accelerate fatty streak formation in LDL-RD mice., Methods and Results: LDL-RD mice were immunized with human beta2GPI. An additional group of mice were immunized with beta2GPI and boosted with the same antigen 3 weeks later. Control mice with immunized with human serum albumin. Lymphocytes obtained from the draining lymph node cells or from splenocytes of beta2GPI- or human serum albumin-immunized mice were stimulated in vitro with beta2GPI or with the mitogen concavalin A, respectively. The cultured lymphocytes were transferred intraperitoneally to syngenic LDL-RD mice, and the mice were fed a high-fat "Western" diet for 5 weeks until death. Mice injected with lymphocytes from draining lymph nodes or spleens of beta2GPI-immunized animals displayed larger fatty streaks than those induced by control treated animals. T-cell-depleted splenocytes from beta2GPI were unable to promote lesion formation in the mice., Conclusions: The present study provides the first direct evidence for a role of antigen (beta2GPI)-reactive T cells in the promotion of fatty streaks in mice.

To study the possible role of the human lipid-oxidizing enzyme 15-lipoxygenase (15-LO) in atherosclerosis, we overexpressed it specifically in the vascular wall of C57B6/SJL mice by using the murine preproendothelin-1 promoter. The mice overexpressing 15-LO were crossbred with low density lipoprotein (LDL) receptor-deficient mice to investigate atherogenesis. High levels of 15-LO were expressed in the atherosclerotic lesion in the double-transgenic mice as assessed by immunohistochemistry. The double-transgenic, 15-LO-overexpressing, LDL receptor-deficient mice (LDLR-/-/15LO) developed significantly larger atherosclerotic lesions at the aortic sinus compared with lesions in the LDL receptor-deficient (LDLR-/-) mice after 3 and 6 weeks (107,000 versus 28,000 microm(2) [P:<0.001] and 121,000 versus 87,000 microm(2) [P:<0.05], respectively) of an atherogenic diet. LDL from the LDLR-/-/15LO mice was more susceptible to oxidation than was the LDL from the control LDLR-/- mice, as shown by a shorter lag period for copper-induced conjugated diene formation. On the other hand, no differences were found in the levels of serum anti-oxidized LDL antibodies between the study groups. There were also no differences with respect to the density of macrophages and T lymphocytes infiltrating the lesions in both experimental groups. Taken together, these results support the hypothesis that 15-LO overexpression in the vessel wall is associated with enhanced atherogenesis.

We have made consecutive studies to prove that autoimmune factors can influence the progression of atherosclerosis in inbred and transgenic mice. C57BL/6 as well as LDL-receptor deficient mice were immunized with heat shock protein 65. LDL-RD and apolipoprotein E knockout (apoE KO) mice were immunized with human B-glycoprotein I. ApoE KO mice were immunized with oxidized LDL. In all immunized mice, a sustained humoral response to the provided antigen was elicited evident by high titers of antibodies by ELISA. A primary cellular immune response was also shown by thymidine incorporation studies employing the antigens in vitro. Immunization with hsp-65 and with beta 2-GPI served to enhance the progression atherosclerosis and led to an increase in the infiltration of CD3 in the subendothelial regions of the early plaques. Transfer of hsp-65 and beta 2-GPI reactive lymphocytes to syngenic mice led to enhancement of fatty streak formation. However, immunization with homologous oxLDL in apoE KO mice led to attenuation of lesion progression concomitant with the production of anti-oxLDL antibodies. Thus, autoimmune factors appear to influence early artherosclerosis progression in mice. If proven in humans these antigen specific responses may be harnessed for selective immunomodulation of the atherosclerotic plaque., (Copyright 2000 Academic Press.)

Anti-endothelial cell antibodies (AECA) have been shown to possess endothelial cell activation properties and to harbor pathogenic potential in experimental animal models of autoimmune systemic disorders. Atherosclerosis is a form of an inflammatory condition in which the immune system has been shown to be involved. The aim of the present study was to assess the presence of AECA in patients with coronary atherosclerosis. A total of 134 patients admitted for chest pain of suspected anginal origin were evaluated for coronary artery atherosclerosis by angiography. Sera were drawn prior to the procedure for the determination of AECA employing cyto-ELISA. AECA positive sera were further evaluated for its ability to promote in vitro E-selectin expression by HUVEC using a cell-based ELISA. Patients with no coronary artery involvement had levels of AECA that did not differ from those obtained for patients with confirmed coronary atherosclerosis (one, two or three vessel disease). Furthermore, AECA positive sera from patients, with or without coronary atherosclerosis displayed similar capacity of inducing E-selectin expression by endothelial cells. AECA may not stand as an optimal mean of discriminating atherosclerotic from non-atherosclerotic patients. The ability of AECA to activate endothelial cells is also not unique to patients with atherosclerosis and is evident also in age-matched control subjects.

Atherosclerotic lesions can be induced in rabbits and mice immunized with heat shock protein 65 (HSP65). In the current study, we investigated the role of interleukin (IL)-4 in the HSP65- and Mycobacterium tuberculosis (MT)-induced models that exhibit an inflammatory phenotype. Fatty streak formation in IL-4-knockout (IL-4 KO) mice immunized with HSP65 or MT was significantly reduced when compared with lesions in wild-type C57BL/6 mice. However, when injected with control (HSP-free) adjuvant, no differences were evident in the lesion size between wild-type and the IL-4 KO mice. Next, we studied comparatively the extent of humoral and cellular immune responses to HSP65 in the IL-4 KO and wild-type mice, as those are thought to be influential in murine atherosclerosis. Anti-HSP65 antibody levels were reduced in the HSP65-immunized IL-4 KO mice as compared with their wild-type littermates, whereas no differences were evident between the groups with respect to the primary cellular immune response to HSP65. Other than the absence of IL-4 in the knockout mice, the pattern of secreting cytokines interferon-gamma and IL-10 in concanavalin A-primed splenocytes was similar between the groups. HSP65-primed inguinal lymphocytes from IL-4 KO mice immunized with HSP65 secreted higher levels of interferon-gamma (previously shown to be proatherogenic in vivo) as compared with their wild-type controls. 12-/15-Lipoxygenase expression, known to be regulated by IL-4 and to contribute to murine atherosclerosis, in the lesions was not influenced by the immunization protocol used or by IL-4 disruption. Thus, IL-4 may prove a principal cytokine in the progression of early "inflammatory" atherosclerotic lesions and may serve as a target for immunomodulation.

We developed a method for the determination of serum 5alpha-cholestan-3beta-ol (cholestanol). The sterols were derivatized to the 4'-bromobenzenesulfonyl esters and heated in isopropanol. The cholesterol-4'-bromobenzenesulfonate was solvolyzed to cholesteryl isopropyl ether, but the derivatized cholestanol did not change and could be measured in a high-performance liquid chromatographic system equipped with a UV detector at 235 nm. On the other hand, the resulting cholesteryl isopropyl ether, having different absorbance and chromatographic mobility was not detected. This method was used for measuring cholestanol levels in patients with cerebrotendinous xanthomatosis (CTX), liver cirrhosis and serum from healthy control subjects. Reproducibility, linearity and recovery tests were done on 0.3 ml of serum samples containing >2 microg/ml cholestanol, using stigmastanol as an internal standard (I.S.). Determining cholestenol by this method can be used for diagnosis and follow-up of patients with CTX and various liver diseases.

Background: Atherosclerosis is a complex histopathologic process that is analogous to chronic inflammatory conditions. Several factors have been shown to correlate with the extent of atherosclerosis. Whereas hypertension, obesity, hyperlipidemia, diabetes, smoking, and family history are all well documented, recent literature points to additional associated factors. Thus, antibodies to oxidized low-density lipoprotein (oxLDL), cytomegalovirus (CMV), Chlamydia pneumonia, Helicobacter pylori, as well as homocysteine and C-reactive protein (CRP) levels have all been implicated as independent markers of accelerated atherosclerosis., Hypothesis: In the current study we attempted to formulate a system by which to predict the extent of coronary atherosclerosis as assessed by angiographic vessel occlusion., Methods: The 81 patients were categorized as having single-, double-, triple-, or no vessel involvement. The clinical data concerning the "classic" risk factors were obtained from clinical records, and sera were drawn from the patients for determination of the various parameters that are thought to be associated with atherosclerosis., Results: Using four artificial neural networks, we have found the most effective parameters predictive of coronary vessel involvement were (in decreasing order of importance) antibodies to oxLDL, to cardiolipin, to CMV, to Chlamydia pneumonia, and to beta 2-glycoprotein I (beta 2GPI). Although important in the prediction of vessel occlusion, hyperlipidemia, hypertension, CRP levels, and diabetes were less accurate., Conclusion: The results of the current study, if reproduced in a larger population, may establish an integrated system based on the creation of artificial neural networks by which to predict the extent of atherosclerosis in a given subject fairly and noninvasively.

Despite numerous studies, the precise role of dietary n-6 polyunsaturated fatty acids in the pathogenesis of atherosclerosis remains controversial. It has been shown that feeding an n-6-enriched diet resulted in decreased atherosclerosis in African green monkeys and was associated with a reduction in LDL levels. However, other authors reported that n-6 supplementation increased the oxidative stress and the susceptibility of LDL to undergo in vitro oxidation, thus potentially enhancing atherosclerosis. The present study was designed to investigate the effect of dietary supplementation of n-6 polyunsaturated fats (safflower oil), as compared with a saturated fat-rich diet (Paigen), on the blood lipid profile and atherosclerosis in two mouse models. In the first experiment, female C57BL/6 mice (n=23-30 per group) were fed a cholate containing Paigen diet, a safflower oil-rich diet (with cholate), or normal chow for 15 weeks. No significant differences between the high fat diet groups were evident with respect to total cholesterol, LDL, HDL or triglyceride levels. The extent of aortic sinus fatty streaks did not differ significantly between the two groups. In the second experiment, LDL-receptor-deficient (LDL-RD) mice (n=20-30 per group) were randomized into similar dietary regimens. Mice consuming a safflower oil-enriched diet developed significantly less atherosclerosis, in comparison with Paigen diet-fed mice. A reduction in LDL levels, although not of a similar magnitude as the reduction in atherosclerosis, was evident in the safflower oil-fed mice when compared to the Paigen diet-fed littermates. In both mouse models of atherosclerosis, LDL isolated from the plasma of mice on the n-6 polyunsaturated diet was rendered slightly more susceptible to oxidation in vitro, as indicated by a shorter lag period for diene formation. Thus, the effects of n-6 fatty acids on the lipoprotein composition and other potential influences may have contributed to the anti-atherogenic effect in the LDL-RD mouse model.

Diabetes and atherosclerosis have been proposed to be influenced by immune and autoimmune mechanisms. A common incriminated antigen in both disorders is the heat shock protein (HSP)-60/65. In the current study, we established a model combining hyperglycemia with hyperlipidemia in LDL receptor-deficient (LDL-RD) mice and assessed its possible influences on lipid profile, HSP60/65, and atherogenesis. LDL-RD mice were injected either with streptozotocin to induce hyperglycemia or with citrate buffer (control). When hyperglycemia was induced, both study groups were challenged with a high-fat (Western) diet for 6 weeks. Plasma fasting glucose, lipid profile, and antibody levels to HSP65 and oxidized LDL were assessed. At death, the spleens from both groups were evaluated for their proliferative response to HSP65 and the consequent cytokine production. The extent of atherosclerosis was assessed at the aortic sinus. Plasma glucose, cholesterol, and triglyceride levels were elevated in mice injected with streptozotocin compared with control mice. Atherosclerotic lesions were significantly larger in the streptozotocin-injected hyperglycemic LDL-RD mice (132 +/- 23 x 10(5) microm2) in comparison to their normoglycemic litter-mates (20 +/- 6.6 x 10(5) microm2; P < 0.0001). Both humoral and cellular immune response to HSP65 was more pronounced in streptozotocin-injected mice. When challenged with HSP65 in vitro, splenocytes from streptozotocin-injected mice favored the production of the T-helper (TH)-1 cytokine gamma-interferon. In conclusion, we have established a mouse model that combines hyperglycemia with diet-induced hyperlipidemia in LDL-RD mice and studied its effect on atherosclerosis progression. The accelerated atherosclerotic process is associated with heightened immune response to HSP65 and a shift to a TH1 cytokine profile.

Heat shock proteins are a family of approximately 25 highly conserved proteins upregulated in response to various forms of stress. They play an active role in the development autoimmune diseases in animals, and have been incriminated in human autoimmune diseases (i.e. rheumatoid arthritis, multiple sclerosis). It has been previously shown, that an induced immune response against Heat shock protein 65 (HSP-65) results in atherosclerotic lesions in normocholesterolemic rabbits. We have supported these findings showing that C57BL/6 mice immunized with HSP-65 and fed a high-fat diet develop enhanced fatty streaks. To create a model that will eliminate the need for exogenous supplementation of a high-fat diet, we have immunized LDL receptor deficient (LDL-RD) mice with HSP-65 or with heat-killed Mycobacterium tuberculosis (Mt). Seven groups of LDL-RD mice (n=10), were immunized subcutaneously with different concentrations of HSP-65, Mt or bovine serum albumin (BSA). All mice were fed a normal chow-diet for 3 months. The mice immunized with the higher doses of Mt developed significantly larger fatty streaks when compared with their BSA immunized littermates. The size of the lesions in the aortic sinus were: 31,562+/-5,994 microm(2)in the 10 microg Mt and 52,777+/-5,245 microm(2)in the 100 microg Mt vs. 11, 500+/-3,750 microm(2)in the BSA group (P<0.05). In the HSP-65-immunized mice, only the group injected with the highest dose (5 microg, twice) developed significantly larger fatty streaks when compared with the BSA-immunized group (28,611+/-4,716 microm(2)vs. 11,500+/-3,750 microm(2)respectively, (P<0.05). The HSP-65-but not the Mt- or BSA-immunized mice developed high titers of anti HSP-65 antibodies, beginning 10 days after the immunization, which persisted until they were killed. Immunohistochemical staining showed CD3-positive lymphocytes in the aortic sinus of mice immunized with Mt or HSP-65, but not in the control group. Thus, we established a mouse model of HSP-65 immune mediated atherosclerosis devoid of high fat diet supplementation. This model will enable us to further study the role of the immune system in atherosclerosis, via HSP-65 and raise novel immunomodulatory therapeutic modalities., (Copyright 2000 Academic Press.)

Objective: Magnesium (Mg) has previously been found to modulate blood lipid levels, atherogenesis and atherosclerosis in rabbits when used as a dietary supplement. In addition, we have reported that Mg fortification of drinking water can attenuate atherogenesis in male low-density lipoprotein (LDL)-receptor-deficient mice, but had a mild and nonsignificant effect on female mice fed a high-cholesterol diet supplemented with cholic acid. The aim of this study was to examine whether Mg has an antiatherogenic effect in female mice fed a high-cholesterol diet without cholic acid., Methods: Two groups of female LDL-receptor-deficient mice were included. The mice received either distilled water or water with 50 g of Mg 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, both without cholic acid. At the end of each stage of the experiment, blood was drawn for the determination of plasma Mg, calcium and lipid levels. In addition, the extent of atherosclerosis was determined at the aortic sinus level., Results: Mg fortification was associated with higher levels of plasma Mg while the mice were on a high-cholesterol diet, and the extent of atherosclerosis at the aortic sinus was significantly decreased in the female mice that received high levels of Mg compared with the female mice that received distilled water. The female mice that received water fortified with Mg had lower levels of triglycerides after stage 2, whereas no differences regarding cholesterol levels were found., Conclusion: These results confirm that Mg fortification of drinking water is capable of inhibiting atherogenesis also in female LDL-receptor-deficient mice fed a high-cholesterol diet, and demonstrate the importance of the nutritional composition of diet in this experimental model., (Copyright 2000 S. Karger AG, Basel)

The modern view of atherosclerosis is of a chronic inflammatory disorder. In accord with this paradigm, the process of uninhibited influx of fat to the vessel wall results from an 'adequate' response to various forms of injury (i.e. turbulence, infections, modified lipoproteins). This idea has been further extended by several groups, to assume that the atherosclerotic lesion can be the target of an autoimmune mediated attack. According to this hypothesis, the site of initiation of the plaque should bear/express the target autoantigen, whereas concomitantly a respective immune response is generated in the periphery. The examples illuminating this notion are beta2GPI as a target autoantigen, HSP60/65 an oxidized-LDL. Herein we present evidence to support the involvement of autoimmune mechanisms in atherogenesis based on the experience from experimental models and human studies.