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We have previously shown that a high cholesterol (HC) diet results in increases in microglia load and levels of the pro-inflammatory cytokine interleukin-6 (IL-6) in the brains of wild type (WT) and apolipoprotein E knockout (ApoE-/-) mice. In the present investigation, we analyzed whether treatment with rosuvastatin, an inhibitor of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, would prevent the increases in inflammatory microglia and IL-6 levels in the brain and plasma of WT and ApoE-/- mice. We report that a HC diet resulted in an increased microglia load in the brains of WT and ApoE-/- mice, in support of our previous study. Treatment with rosuvastatin significantly decreased the microglia load in the brains of WT and ApoE-/- mice on a HC diet. Rosuvastatin treatment resulted in lowered plasma IL-6 levels in WT mice on a HC diet. However, in the present study the number of IL-6 positive cells in the brain was not significantly affected by a HC diet. A recent clinical study has shown that rosuvastatin reduces risk of ischemic stroke in patients with high plasma levels of the inflammatory marker C-reactive protein by 50%. The results from our study show that rosuvastatin reduces inflammatory cells in the brain. This finding is essential for furthering the prevention and treatment of neurodegenerative diseases such as Alzheimer's disease (AD) and stroke.

Background and Purpuse: Cerebral White matter changes (WMC) are a frequent finding on CT and MRI scans of elderly individuals, particularly in those with vascular risk factors, cerberovascular disease, and cognitive impairment. Methods: 56 subjects were included in the study after the review of reports of more than 200 consecutive brain Computerized Tomography (CT) and magnetic resonance imaging (MRI) examinations from the out-patient and in-patient units of the Department of Geriatric Medicine at Karolinska University Hospital, Huddinge during 2001-2002. MRI was performed using a 1.5 T system and WMC lesions were graded 1-3 using a visual scale. Total-cholesterol (TC), high density lipoprotein (HDL), low density lipoprotein (LDL) and triglyceride (TG) levels were determined using enzymatic techniques after 12 hours overnight fasting. Apo E genotyping was performed as described. Results: Low HDL levels were associated with higher severity of WMC on MRI (p=0.002). Subjects with the Apo E4 allele had higher LDL (p=0.002) and apoB levels (p=0.005). The presence of the Apo E4 allele was higher in the group of subjects with severe WMC (grade 3). However, there was no statistically significant group difference in severity of WMC lesions between carriers and noncarriers of Apo E4 allele. Conclusions: Low HDL is strongly associated with adverse coronary and cerebrovascular outcomes. Our results indicate that low HDL levels are also associated with more severe WMC lesions on MRI. Dietary or medical adjustment of HDL levels could have important implications for treatment and prevention of cerebral WMC, cerebrovascular and neurodegenerative diseases such as stroke and dementia.

The processing of the Amyloid Precursor Protein (APP) is a critical event in the formation of amyloid plaques which are composed of the 4kDa amyloid beta-peptide (Abeta). Processing of APP occurs through a non-amyloidogenic pathway, mediated by initial alpha-secretase cleavage or through an amyloidogenic pathway via sequential cleavage by beta- and gamma-secretase enzymes, which produces Abeta peptides. Currently, the diagnosis of probable or possible Alzheimer's disease (AD) is primarily based on neuropsychological and neuroradiological assessment. Recent reports indicate that platelet beta-secretase activity is moderately increased in patients with AD and mild cognitive impairment (MCI). To our knowledge platelet alpha-secretase activity has not yet been explored in this context and estimation of the ratio of the activities of alpha- and beta-secretase in platelets may represent a useful surrogate marker of the balance between the two pathways of APP metabolism and be of importance for the diagnosis of AD. We therefore considered it of interest to develop assays of platelet alpha- and beta-secretase activities suitable for such clinical investigations. Application of these assays to a Swedish population failed to uncover an effect of AD or MCI on individual platelet secretase activities or the secretase ratio. However, we did observe an inverse correlation between plasma triacylglycerol levels and the secretase ratio. The results are discussed in the context of the clinical usefulness of the secretase ratio as a biochemical adjunct to the diagnosis of AD.

In order to reveal the neuroprotective effects of statins that could be of interest for the prevention and treatment of Alzheimer's disease (AD), we investigated the expression of nicotinic acetylcholine receptors (nAChRs) detected by RT-PCR, the activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) by colorimetric determination, and the levels of the alpha-form of secreted beta-amyloid precursor protein (alphaAPPs) by Western blotting in neuroblastoma (SH-SY5Y) cells exposed to lovastatin, atorvastatin, rosuvastatin and simvastatin, respectively. The results indicated that all statins studied, both lipophilic and hydrophilic, induced high expression of alpha7 nAChR, decreased cholinesterase activities, and increased alphaAPPs, which suggests that statins might play important neuroprotective roles in AD treatment.

The influence of a high cholesterol (HC) diet on brain pathology is being recognized increasingly and is of immense interest. Previous findings from our laboratory demonstrated that a high cholesterol diet increases gliosis, astrocytic reactivity and neuroinflammation in both wild type (WT) and apolipoprotein knockout (ApoE-/-) mice. In the present study, we analyzed whether this increase in astrocytic reactivity, monitored by the number of cells in the hippocampus labelled with glial fibrillary acidic protein (GFAP), could be reduced by the use of rosuvastatin, a potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase. Furthermore, we studied the effect of rosuvastatin on changes in lipoprotein levels and weight gain, and their correlation to gliosis, in mice fed a high cholesterol diet. A significant increase in weight, total-cholesterol (TC) and low-density lipoprotein (LDL) levels were observed in WT and ApoE-/- mice on a HC diet. The number of GFAP labelled cells was found to be significantly increased in mice on a HC diet and reduced in rosuvastatin-treated WT and ApoE-/- mice on a HC diet. A significant reduction of weight, total-cholesterol and LDL levels was observed in rosuvastatin-treated WTHC mice. Significant correlations were found between changes in body weight, GFAP labelled cells and plasma total-cholesterol levels in WT and ApoE-/- mice. However, the correlations were found to be weaker for the GFAP labelled cells in the ApoE-/- mice. The results indicate that the observed reduction of gliosis by rosuvastatin treatment may be due to mechanisms that are independent of its lipid-lowering effect.

Hypercholesterolemia has been suggested as a risk factor for Alzheimer's disease (AD). A genetic risk factor for AD is the E4 allele of apolipoprotein E (apoE). ApoE is the major lipoprotein transporter in the brain, and is mainly produced by glial cells. The present study is focussed on analysing the effects of high cholesterol (HC) diet, duration 9 months, on glial activation in the brain, both in wild type (WT) mice and in mice with a null mutation in the apoE gene (knock-out, KO) mice. The activation of astrocytes and microglia was analysed after immunohistochemical labelling of glial fibrillary acidic protein (GFAP), and F4/80, respectively. In addition, the expression of the antioxidant enzyme NAD(P)H:quinone oxidoreductase (NQO1) was analysed. There was a marked stimulation of astrocyte and microglial activation as well as induced expression of NQO1 in the hippocampus and cerebral cortex upon HC diet. Furthermore, there was significant astrocyte activation in the apoE KO mice, as compared to the WT mice, on ND. The long time exposure to HC diet combined with apoE deficiency resulted in a synergistic effect on the expression of NQO1 in the brain.

alpha1-Antitrypsin (AAT) serine proteinase inhibitor is found in most biological fluids, diffuses into most tissues, and is an important factor in controlling tissue damage by proteases in inflammatory diseases such as atherosclerosis. We have previously reported that the C-terminal fragment (C-36) generated during the cleavage of AAT by proteinases forms amyloid fibrils which have biological effects unrelated to precursor functions. Here we show that the C-36 fragment is present in atherosclerotic plaques, particularly within the fibrous cap at the base of the lipid core. We also found that human monocyte stimulation with C-36 fibrils led to a strong activation of both peroxisome proliferator-activated receptors alpha and gamma (PPARalpha and PPARgamma) at 1, 2, and 18 h of cell culture. A parallel increase in the intracellular lipid accumulation was also observed. Furthermore, stimulation of monocytes with C-36 for 18 h led to activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) activation. These data for the first time demonstrate the peptide of AAT as a component of atherosclerotic plaques and as a novel activator of PPARalpha, PPARgamma, NF-kappaB, and AP-1 in cultured monocytes. Taken together, the effects of the peptide represent a new mechanism of monocyte activation that may be of importance not only in atherogenesis, but also in other inflammatory processes.

Background Ischaemic cerebrovascular disease (ICVD) is a heterogeneous syndrome to which different genetic factors may contribute. We have investigated the distribution of alleles of the angiotensin-converting enzyme (ACE) gene, which has been suggested to be of possible importance in ischaemic stroke or cardiovascular disease, in groups of patients with ischaemic stroke and carotid artery stenosis (CS). Materials and methods One hundred and thirty patients with ischaemic stroke and 68 patients with more than 50% stenosis of the internal carotid artery were investigated and compared with age- and sex-matched healthy control subjects. Alleles of an insertion/deletion polymorphism of the ACE gene were determined by one-stage polymerase chain reaction and visualized on agarose gels. Results There was a significant difference (P

Methods A biallelic polymorphism of the methylenetretrahydrofolate reductase (MTHFR) gene, reported to influence the plasma level of homocysteine (Hcy), was investigated for a possible role in influencing the risk of ischaemic cerebrovascular disease (ICVD) and occlusive atherosclerosis in 126 patients with ischaemic stroke and 70 patients with internal carotid artery (ICA) stenosis. Results Only minor differences were observed between different groups of patients and control subjects. Although 47% of ICA stenosis patients had increased plasma Hcy, the MTHFR genotype did not correlate with levels of either Hcy, folic acid or vitamin B12. In addition, the MTHFR genotype did not affect Hcy levels, even in the presence of low blood folate. Conclusion We conclude that this common MTHFR gene polymorphism does not exert a significant influence on the risk of developing ICVD or ICA stenosis, and does not cause the increased level of Hcy observed in ICA stenosis.

We determined whether plasma levels of circulating oxidized low-density lipoprotein (LDL; E06), immunoglobulin (Ig) G, and IgM autoantibodies binding to malonyldialdehyde-modified LDL (MDA-LDL) may predict cardiovascular events (CVEs). Patients (n = 123) with a previous myocardial infarction (MI) were included. The primary end point was defined as any of the following: cardiovascular death from any cause, nonfatal reinfarction or stroke, percutaneous coronary intervention, coronary artery bypass grafting, and hospitalization due to angina pectoris. There were 43 CVEs during the follow-up period of 8.4 ± 3.5 years. There was no significant difference in the levels of E06 and MDA-LDL IgG between the CVE and the event-free group. However, MDA-LDL IgM levels were significantly lower in patients in the CVE group (9524 ± 6326 relative light unit [RLU]) compared with the event-free (10 975 ± 5398 RLU) group ( P = .04). In conclusion, levels of MDA-LDL IgM were associated with an increased risk of CVE in patients with a previous MI.

The association between C-reactive protein (CRP) and future cardiovascular risk has been of particular interest during recent years. Oxidized low-density lipoprotein (ox-LDL) is another marker linking the immune system with the atherogenic process. The aim of this study was to examine whether ox-LDL and CRP were associated with endothelial function in peripheral resistance arteries. Twenty-five men with a previous hospital-diagnosed myocardial infarction were enrolled in the study. The exclusion criterion was a history of diabetes mellitus. IgG and IgM autoantibodies to malonyldialdehyde low-density lipoprotein (MDA-LDL) and high-sensitivity CRP (hs-CRP) were measured. Flow-mediated dilatation was measured in isolated resistance arteries from subcutaneous fat biopsies. Endothelial function test reflecting the maximum vessel dilatation in male subjects was inversely related to MDA-LDL IgG autoantibody levels (r = -0.6 and P = 0.003). Comparison of hs-CRP levels and of maximum vessel dilatation in males revealed also an inverse relation (r= -0.4 and P = 0.04). In conclusion, a clear correlation exists between flow-mediated dilatation in subcutaneous resistance arteries and plasma levels of MDA-LDL IgG autoantibody and CRP in male patients with coronary heart disease.

The cholesterol 24-hydroxylase encoded by the gene CYP46 is expressed almost exclusively in central nervous system (CNS) neurons and catalyzes the formation of 24S-hydroxycholesterol (24S-OHC) from cholesterol. This conversion corresponds to a major pathway for excretion of excess cholesterol from the brain. There is a significant flux of another oxysterol, 27-hydroxycholesterol (27-OHC) from the circulation into the brain. Polymorphisms within the CYP46A1 gene have been associated with Alzheimer's disease (AD) incidence. In this study, we examined the effects of 24S-OHC and 27-OHC on the alpha- and beta-secretase activity in the human neuroblastoma cell line SH-SY5Y. Furthermore, we examined the effects of the two oxysterols on the levels of extra- and intracellular proteins of secreted APPalpha (sAPPalpha). Our findings suggest that 24S-OHC may exert a unique modulatory effect on APP processing and that this oxysterol increases the alpha-secretase activity as well as the alpha/beta-secretase activity ratio. The possibility is discussed that the ratio between 24S-OHC and 27-OHC is of importance for the generation of amyloid in the brain.

Inflammation in the central nervous system is an early hallmark of many neurodegenerative diseases including Alzheimer's disease (AD). Recently, increasing evidence suggests that hypercholesterolemia during midlife and abnormalities in the cholesterol metabolism could have an important role in the pathogenesis of AD. In the present study, we have evaluated the effect of high cholesterol (HC) diet on the expression of interleukin-6 (IL-6), a cytokine involved in neurodegeneration, and caspase-1, that is responsible for the cleavage of the precursors of interleukin-1 beta (IL-1 beta) and interleukin-18 (IL-18) in the brain of apolipoprotein E (Apo E) knock-out (KO) and wild type (WT) mice. The density of IL-6-positive cells was increased in the hippocampus (p

We analysed the effects of high cholesterol (HC) intake and reduced apolipoprotein E (apoE) activity on tau phosphorylation and on the activities of the major tau kinases and phosphatases in brains from wild-type and apoE-knockout (apoEKO) mice. We show that HC diet potently induced intraneuronal accumulation of hyperphosphorylated tau in apoEKO mice, as well as upregulation of several tau kinases, without affecting tau phosphatases. Our results suggest an interaction between dietary and genetic factors in the development of tauopathies, which can be relevant in humans, where the apoE4 isoform could have a lack of function as compared to other isoforms.

3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors, statins, are widely used cholesterol-lowering drugs and have been shown to have anticancer effects in many models. We have investigated the effect of statins on Mdm2, a p53-specific ubiquitin ligase. It was found that pravastatin induced Mdm2 phosphorylation at Ser166 and at 2A10 antibody-specific epitopes in HepG2 cells, while mRNA levels were unchanged. Furthermore, pravastatin was found to induce phosphorylation of mTOR at Ser2448. Ser166 phosphorylation of Mdm2 was abrogated by an inhibitor of mTOR, rapamycin, but not by the PI3-kinase inhibitors LY294002 and wortmannin. Ser166 phosphorylation of Mdm2 has been associated to active Mdm2 and has been shown to increase its ubiquitin ligase activity and lead to increased p53 degradation. Our data show that statins attenuated the p53 response to DNA damage. Thus, in HepG2 cells pravastatin and simvastatin pretreatment attenuated the p53 response to DNA damage induced by 5-fluorouracil and benzo(a)pyrene. Similar attenuation was induced when p53 stabilization was induced by the inhibitor of nuclear export, leptomycin B. Furthermore, in the DNA-damaged cells, half-lives of Mdm2 and p53 were decreased by statins, indicating a more rapid formation of p53/Mdm2 complexes and facilitated p53 degradation. The induction of p53 responsive genes and apoptosis was attenuated. Mdm2 and p53 were also studied in vivo in rat liver employing immunohistochemistry, and it was found that constitutive Mdm2 expression was changed in livers of pravastatin-treated rats. We also show that the p53 response to a challenging dose of diethylnitrosamine was attenuated in hepatocytes in situ and in primary cultures of hepatocytes by pravastatin pretreatment. Taken together, these data indicate that statins induce an mTOR-dependent Ser166 phosphorylation of Mdm2, and this effect may attenuate the duration and intensity of the p53 response to DNA damage in hepatocytes.

An increasing number of studies suggest that disturbances in cholesterol homeostasis may promote the formation and deposition of beta-amyloid (A beta) and the progression of Alzheimer's disease (AD). In this paper, we have analyzed the effect of the lipid-lowering compound rosuvastatin on apoptosis and caspase-3 activity in human neuroblastoma SH-SY5Y cells. Exposure of SH-SY5Y cells to A beta(1-42) alone resulted in a significantly increased caspase-3 activity approximately by 35% (135+/-15%, p

Abstract Degeneration of smooth muscle cells in the fibrous cap of atherosclerotic lesions is an important factor in plaque rupture. Recent studies have suggested that many plaque cells are in a process of apoptosis as determined by positive deoxyribonucleotide-transferase-mediated dUTP end labeling. In this study, we demonstrate the existence of a colocalization between deoxyribonucleotide-transferase-mediated dUTP end labeling-positive smooth muscle cells and oxidized LDL immunoreactivity in human carotid plaques. Oxidized LDL was found to induce deoxyribonucleotide-transferase-mediated dUTP end labeling positivity in cultured human smooth muscle cells, but only in the presence of tumor necrosis factor-α and interferon-γ. Electron microscopic analysis of cultured smooth muscle cells exposed to oxidized LDL in the absence of cytokines demonstrated cytoplasmic swelling and disruption of the plasma membrane, suggesting cell death by oncosis. Cells exposed to both oxidized LDL and cytokines were characterized by chromatin and cytoplasmic condensation compatible with cell death by apoptosis. These findings further support the notion that oxidized lipids play a role in plaque cell death.

The aim of the present study was to analyze the frequency and mechanism of cell death in atherosclerotic plaques with a recent history ( 70% diameter reduction) undergoing carotid endarterectomy. In situ tailing nick translation of fragmented DNA, agarose gel electrophoresis of plaque DNA and electron microscopy were used to identify cell death by apoptosis (programmed cell death) and necrosis. The mean number of cell containing fragmented DNA in the plaques was 12.7 ± 3.5% (n=15). Focal accumulations of cells with DNA fragmentation occurred in the fibrous cap, at sites of rupture, close to lipid deposits and necrosis and was always accompanied by the presence of inflammatory cells. Electrophoretic separation of DNA isolated form part of plaques where the presence of DNA fragmentation had previously been demonstrated by in situ DNA nick translation resulted in multiple ladders of 180–200 base pairs characteristic of apoptosis. Electron microscopic analysis revealed presence of cells with morphological signs of degeneration in a frequency even higher than that found by in situ nick translation. Some of these cells had a characteristic apoptotic appearance with condensed chromatin and cytoplasm, but the large majority of the cells had an ultrastructure typical for cells undergoing cell death by necrosis with membrane disruption and swollen, disintegrating organelles. Thus, although apoptosis clearly takes place in atherosclerotic plaques necrosis appears to be a much more common mechanism for cell death. This may have negative consequences for plaque stability since cell death by necrosis induces a stronger inflammatory reaction than apoptotic cell death.

It has recently been shown that extrahepatic cells can eliminate intracellular cholesterol by enzymatic conversion into 27-hydroxy-cholesterol and 3 beta-hydroxy-5-cholestenoic acid. Using immunohistochemical methods, we studied the presence of the enzyme responsible for these conversions, sterol 27-hydroxylase, in human carotid atherosclerotic plaques. All plaques examined were found to contain sterol 27-hydroxylase immuno-reactive cells. While some endothelial cells stained for sterol 27-hydroxylase, the majority of the immunoreactive cells co-localized with macrophages. Accumulation of sterol 27-hydroxylase-positive cells were often observed in macrophage-rich core regions of complicated lesions. High concentrations of 27-hydroxycholesterol were found in plaques, while the concentration in non-atherosclerotic human vessels was lower by two orders of magnitude. The rabbit, which is particularly sensitive to dietary cholesterol and easily develops fatty streaks, had low plasma levels of 27-hydroxycholesterol, 3 ng/ml compared to 150 ng/ml in humans. The concentration of 27-hydroxycholesterol in the atherosclerotic rabbit vessels was also lower compared to human atherosclerotic plaques. The results are consistent with our hypothesis that sterol 27-hydroxylase may be utilized by human macrophages as a defence towards a high cholesterol load. This mechanism may be less important in some other species.

Statins reduce cholesterol levels through competitive inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the key enzyme that regulates cholesterol synthesis. The cholesterol-lowering effect of statins is also due to an increase in the uptake of cholesterol by cells as a result of intracellular cholesterol depletion and enhanced expression of low-density lipoprotein (LDL) receptors. The use of statins as lipid-lowering agents has lead to remarkable changes in the treatment and prevention of ischemic heart disease. Results of large clinical trials of patients with ischemic heart disease have demonstrated that statins reduce inflammatory markers such as C-reactive protein, an independent risk factor in the disease. Statins exhibit properties that are beyond their lipid-lowering effects. These non-lipid-lowering properties involve the inhibition of the isoprenoid pathway through decreased synthesis of many nonsteroidal isoprenoid compounds. The focus on the immunomodulatory effect of statins is the result of the positive outcome of pravastatin treatment in cardiac transplantation patients, as well as angiographic regression studies showing insignificant changes in the degree of coronary stenosis despite a large reduction in cardiac events. Statin treatment reduces the risk of ischemic stroke despite the fact that LDL cholesterol is not directly associated with the risk of stroke. This observation lead to the investigation of the role of statins in inflammation and the immune system. Recent research data demonstrated that statins inhibit the induction of the major histocompatibility (MHC) class II expression by interferon-gamma (IFN-gamma), leading to repression of MHC II-mediated T-cell activation. Furthermore, statins inhibit the expression of specific cell surface receptors on monocytes, adhesion molecules and also integrin-dependent leucocyte adhesion. While statins may stimulate the secretion of caspase-1, IL-1beta and IL-18 in peripheral mononuclear cells in response to Mycobacterium tuberculosis, they exhibit additional effects on inflammation by decreasing IL-6 synthesis in human vascular smooth muscle cells (VSMC) in vitro. The focus of this monograph is to highlight the role of statins in the modulation of the immune system and inflammatory processes.