Elevated low-density lipoprotein (LDL) concentration in mid-life increases the risk of developing Alzheimer's disease (AD) in later life. Increased oxidized LDL (oxLDL) modification and nitration is observed during dementia and hypercholesterolaemia. We investigated the hypothesis that statin intervention in mid-life mitigates the inflammatory effects of oxLDL on the microvasculature. Human microvascular endothelial cells (HMVECs) were maintained in transwells to mimic the microvasculature and exposed to patient and control LDL. Blood was obtained from statin-naive, normo- and hyper-lipidaemic subjects, AD with vascular dementia (AD-plus) and AD subjects (n=10/group) at baseline. Only hyperlipidaemic subjects with normal cognitive function received 40 mg of simvastatin intervention/day for 3 months. Blood was re-analysed from normo- and hyper-lipidaemic subjects after 3 months. LDL isolated from statin-naive hyperlipidaemic, AD and AD-plus subjects was more oxidized (agarose gel electrophoretic mobility, protein carbonyl content and 8-isoprostane F2α) compared with control subjects. Statin intervention decreased protein carbonyls (2.5±0.4 compared with 3.95±0.2 nmol/mg; P<0.001) and 8-isoprostane F2α (30.4±4.0 pg/ml compared with 43.5±8.42 pg/ml; P<0.05). HMVEC treatment with LDL-lipids (LDL-L) from hyperlipidaemic, AD and AD-plus subjects impaired endothelial tight junction expression and decreased total glutathione levels (AD; 18.61±1.3, AD-plus; 16.5±0.7 nmol/mg of protein) compared with untreated cells (23.8±1.2 compared with nmol/mg of protein). Basolateral interleukin (IL)-6 secretion was increased by LDL-L from hyperlipidaemic (78.4±1.9 pg/ml), AD (63.2±5.9 pg/ml) and AD-plus (80.8±0.9 pg/ml) groups compared with healthy subject lipids (18.6±3.6 pg/ml). LDL-L isolated after statin intervention did not affect endothelial function. In summary, LDL-L from hypercholesterolaemic, AD and AD-plus patients are inflammatory to HMVECs. In vivo intervention with statins reduces the damaging effects of LDL-L on HMVECs.

CLINICAL PERSPECTIVES

  • We have explored why patients who have been prescribed lipid-lowering drugs in mid-life are at a lower risk of dementia. We identified a novel anti-inflammatory effect for statins in patients with hypercholesterolaemia, by preventing LDL-lipid oxidation.

  • The inflammatory and oxidizing effects of LDL fractions towards microvascular endothelial cells (measured as directional cytokine secretion and cellular glutathione) were greatest for oxLDL-L and then LDL-L with oxLDL-protein being the least inflammatory. Moreover, in common with adults with hypercholesterolemia, LDL from AD-plus and AD patients was more oxidized, elicited more endothelial glutathione depletion, more IL-6 secretion and impaired endothelial tight junctions.

  • Thus, the mechanisms triggering LDL oxidation and the ability of oxidized LDL to endothelial dysfunction could be targeted by statins to reduce the risk for vascular involvement and dementia development.

INTRODUCTION

In the adult brain, primary cholesterol synthesis occurs in astrocytes and to a lesser extent in neurons [1]. Cholesterol is transported within the brain by local lipoproteins. Brain cholesterol metabolism is discrete from metabolism in peripheral tissues and the central nervous system (CNS) and plasma cholesterol/lipoprotein compartments are strictly segregated by the blood–brain barrier (BBB).

The BBB is created by the tight junctions between the endothelial cells of brain microvascular tissue assisted by the astrocyte foot processes surrounding the capillary endothelial cells [2,3]. Transfer across the BBB to the CNS is achieved via ATP-dependent transporters, facilitated diffusion, transmembrane diffusion and leakage through extracellular pathways.

There is no evidence that normal lipoprotein cholesterol [e.g. high-density lipoprotein-cholesterol (HDL-C) and low-density lipoprotein-cholesterol (LDL-C)] originating in the plasma compartment routinely crosses the BBB to be transported into the CNS [4]. In support of this, knockout of the low-density lipoprotein (LDL) receptor gene in mice and rabbits or disruption of peripheral scavenger receptor BI (SR-BI) or ATP-binding cassette receptor A1 (ABCA1) in mice changes neither the cholesterol synthesis rate nor the cholesterol concentration in the brain [5,6]. Taken together, these data are in agreement with the view that plasma cholesterol is not transported to the brain because plasma lipoproteins do not cross the BBB. Nevertheless, prospective studies have shown a reduced risk of Alzheimer's disease (AD) in users of cholesterol-lowering statin drugs [7]. Many previous studies have demonstrated the beneficial effects of statins on endothelial dysfunction and chronic inflammation [8,9]. In addition, an association between previous statin use and reduced neurofibrillary tangle burden post-mortem has also been reported [10,11]. In the transgenic CRND8 (TgCRND8) mouse model of AD, a reduction in β-amyloid accumulation was been observed in the brain after treatment with pravastatin, a non-hydrophilic non-BBB-permeant statin [12], supporting the hypothesis that peripheral cholesterol metabolism is important to brain pathology.

Hypercholesterolaemia is an important contributor to oxidative stress [13], a condition of imbalance between oxygen free radical production and removal by antioxidant enzymes and micronutrients. If radical production exceeds that which can be controlled by antioxidants, irreversible damage to proteins, lipids and DNA occurs. Evidence of oxidation and nitration of proteins to form carbonyl groups and 3-nitrotyrosine is frequent in plasma from both hypercholesterolaemic and dementia patients [14,15]. Similarly, free-radical-oxidized lipids (e.g. isoprostanes, lipid hydroperoxides) are more prevalent in plasma from AD and hypercholesterolaemic subjects compared with controls [16]. Together, these studies implicate systemic lipid/protein oxidation as a common feature of AD and hypercholesterolaemia. Although the beneficial effects of statins against cardiovascular disease in the latter group have been attributed to reduction in cholesterol, previous studies suggest that benefit is due to oxidized LDL (oxLDL) removal [17]. Indeed, clinical trials showing protective effects of statins against AD do not show that a reduction in plasma cholesterol correlates with reduced risk of AD, indicating that an alternative mechanism for the beneficial effects of statins may be present [18]. It is not known whether removal of oxLDL accounts for the protective effects of statin usage in mid-life against the later AD development.

Therefore, we have examined the hypothesis that systemic oxLDL affects the function of microvascular endothelial cells to secrete inflammatory mediators into the brain compartment pre-disposing to AD and that statin intervention renders LDL less inflammatory. We have undertaken qualitative and quantitative analysis of lipid oxidation products in LDL isolated from mid-life subjects with elevated plasma cholesterol, investigated whether statin intervention in hypercholesterolaemic subjects can prevent modifications to LDL in vivo and whether any protection of LDL oxidation restores normal microvascular endothelial cell function.

MATERIALS AND METHODS

Plasma sample preparation

Twenty mid-life male adults (40–60 years old, mean age 46.9 years) were recruited from general medical practices in the Birmingham area with (total cholesterol >6.5 mM measured) and without hypercholesterolaemia. All hypercholesterolaemic subjects were unresponsive to lifestyle change, were statin-naive and were not taking any disease-modifying anti-inflammatory medication or nutritional supplements. All subjects were healthy by careful clinical history and examination and scored at least 27 on the mini-mental state examination. The research has been carried out in accordance with the Declaration of Helsinki (2008) of the World Medical Association and ethical approval was obtained from the Birmingham and Black Country Local Research Ethics Committee (REC 09/H1202/87). Participants provided informed written consent.

After an overnight fast, 5 ml of whole blood was drawn from the antecubital vein of each participant and collected into EDTA-coated tubes (Greiner Bio-One Ltd) between 8:00 a.m. and 10:30 a.m. as a baseline measure. All ten statin-naive hypercholesterolaemic subjects were started on simvastatin intervention (40 mg/day) as a revision to their routine management after lifestyle changes had failed to reduce blood cholesterol, whereas normolipidaemic subjects maintained habitual diets and lifestyles without intervention. According to National Institute for Health and Care Excellence (NICE) guidelines (http:://www.nice.org.uk/guidance/cg181), patients were recruited after 3 months to a second blood sampling. All ten hypercholesterolaemic patients completed the intervention for the study duration of 3 months. AD subjects were recruited from the Unit of Cognitive Frailty, Neurology Outpatient Clinic, Cologne, Germany, after diagnosis of AD using National Institute of Neurological and Communicative Disorders and Stroke and Alzheimer's Disease and Related Disorders Association (NINCDS–ADRDA) criteria either in the presence of vascular comorbidities and risk factors [high cholesterol, hypertension, stroke, Type 2 diabetes mellitus, myocardial infarction and heart arrhythmia; AD with vascular dementia (AD-plus) group] or without (AD group) cardiovascular comorbidities and risk factors [19]. Three patients in the AD group were diagnosed with hypertension alone. After an overnight fast, 5 ml of whole blood was drawn from the antecubital vein of each participant and collected into EDTA. Informed consent was obtained from the patients or their care givers according to severity of disease and the study was approved by the local ethics committee. Blood plasma was prepared by centrifugation (200 g, 30 min) and stored immediately at–80°C prior to analysis and extraction of LDL.

Isolation, modification, characterization of LDL and LDL-lipids

From all blood samples obtained, LDL was isolated, modified and characterized as described previously [20]. LDL protein concentration was analysed by BCA assay (Sigma–Aldrich). In addition, LDL lipid content was analysed by Amplex® Red cholesterol assay kit (Life Technologies) and phospholipid assay. The degree of lipid oxidation of LDL and oxLDL was determined as 8-isoprostane F2α levels (15.5±1 pg/mg of LDL and 26±2.5 pg/mg of oxLDL) using an enzyme immunoassay assay method according to the manufacturer's instructions (Cayman Chemicals). LDL-lipids (LDL-L) or oxLDL-lipids (oxLDL-L) were extracted from LDL using the Folch method by the addition of 160 μl of ice-cold methanol (containing 50 μg/ml butylated hydroxytoluene, BHT) followed by the addition of 320 μl of ice-cold chloroform and incubation for 20 min on ice with occasional vortex-mixing. High-purity water (150 μl) was added and the sample was kept on ice for an additional 10 min with occasional mixing. The sample was centrifuged for 5 min at 200 g and the upper (aqueous) phase was removed and re-extracted by the addition of 250 μl of ice-cold chloroform/methanol (2:1, v/v) as above. The upper phase was discarded and both organic phases were combined, dried under nitrogen gas and kept at–80°C until further use. Extracted lipids for cell culture experiments were conjugated to fatty acid-free BSA in serum-free Roswell Park Memorial Institute (RPMI) 1640 medium [25].

Phospholipid assay

Total phospholipid content in human microvascular endothelial cell (HMVEC) lysates were analysed as described previously with some modifications [21]. A phospholipid standard curve (0.005–0.05 mg/ml egg yolk lecithin) was prepared by diluting 0.1 mg/ml stock solution in chloroform in glass vials. One millilitre of 0.1 M ammonium ferrothiocyanate [2.7 g of ferric chloride hexahydrate (FeC13·6H2O) and 3.04 g of ammonium thiocyanate (NH4SCN) in deionized distilled water and made up to 100 ml] was added to 1 ml of lipid standards or sample and vortex-mixed for 1 min. The layers were allowed to separate for 5 min before removing chloroform layer into a new glass tube. Samples and standards were read using a spectrophotometer at 488 nm in a quartz cuvette.

Agarose gel electrophoresis

Lipoprotein was assessed by 1% agarose gel electrophoresis in barbital buffer as described previously [22]. Briefly, Bromophenol Blue in glycerol (50%; 4 μl) was added to LDL samples (20 μg) before electrophoresis in barbital buffer (sodium barbital 10.6 g/l, pH 8.6) for 2 h at 55 V. Gels were stained with Coomassie Brilliant Blue to visualize proteins for approximately 45 min. The gel was destained (40% methanol, 10% acetic acid and 50% water), for 30 min, prior to exchange for fresh destain and then left overnight. Relative mobility (RF) values were calculated for each sample as the distance the sample moved from origin (mm) divided by the distance travelled by the dye front (mm).

Microvascular endothelial model

HMVECs (Invitrogen, Life Technologies) were seeded (3×105/ml) on to 24-well polycarbonate inserts and cultured for 7 days. LDL-L or oxLDL-L (0.4–8 μg) or control/patient LDL-L (4 μg) was added on to endothelial barrier for 24 h before measuring the change in barrier permeability after treatment with 200 μl of 1 mg/ml FITC–dextran apically for 24 h. The basolateral and apical media were collected, the apical surface was washed twice, and the washes were collected and combined with the apical medium collected. Fluorescence of the media was read with excitation at 488 nm and emission wavelength 520 nm using a Spectra Max Gemini XS fluorimeter (Molecular Devices). The percentage fluorescence in the basolateral compartment was calculated as a measure of cell permeability.

ELISA

Following the cell treatments with lipids, cell culture media were collected and cells were pelleted by centrifugation (200 g, 10 min), cell-free medium containing secreted cytokines was stored at–20°C until analysis for interleukin (IL)-6 and tumour necrosis factor (TNF)α by ELISA (Peprotech).

Cell viability assay

Cell viability was measured using the Cell Titer-Blue® viability assay. After incubation with 100 μl of Cell Titer-Blue®, supernatants from Transwell® inserts were removed to a 96-well plate and fluorescence was measured with excitation at 560 nm and emission at 590 nm using a Spectra Max Gemini XS fluorimeter (Molecular Devices).

Zonula occludens-1 staining

HMVECs were seeded on to Transwell inserts and, after 7 days, cells were fixed with 100% methanol (pre-cooled) prior to permeabilization with 0.1% (v/v) Triton X-100. Cells were then blocked with 100 μl of 1% (v/v) normal goat serum before incubating with mouse monoclonal anti-ZO-1 (zonula occludens-1) antibody diluted 1:250 in 1% BSA overnight at 4°C. Cells were then stained with goat anti-mouse FITC-conjugated secondary antibody diluted 1:100 in 1% BSA and mounted with hard set mounting medium containing DAPI for 48 h at 4°C in the dark. Images were visualized using a Zeiss LSM 510 META confocal laser-scanning microscope with a × 63 oil objective, a green filter cube (495nm) and a blue filter cube (365 nm) for visualization. Images were analysed using LAS AD Lite software (Leica Microsystems).

Glutathione (GSH) assay

HMVEC GSH levels were measured using a GSH–Glo assay (Promega).

Lipid peroxidation analysis

Plasma 8-isoprostane F2α was measured by ELISA (Cayman Chemicals). This kit provided inter-assay variation (%CV) of 16.6 and intra-assay variation (%CV) of 16.2. A lower limit of detection was 20 pg/ml.

Statistical analysis

Statistical significance was tested by using ANOVA with Tukey's post-hoc test, Student's t test or Wilcoxon's matched paired t test using Prism 6 (GraphPad). Unless specified all data are presented as the means ± S.E.M. for at least three independent experiments, each performed in triplicate.

RESULTS

LDL and oxLDL-lipids increase microvascular endothelial permeability in vitro

Previously we have shown that LDL and its lipids are more oxidized in dementia; therefore, we examined the effects of LDL and LDL-L that had been isolated from patients with hypercholesterolaemia or dementia and their respective controls directly on HMVECs.

HMVECs were cultured on Transwells until a barrier was formed with ZO-1 expression localized to membrane junctions at day 7. Monolayers were subsequently exposed to control LDL from healthy donors with and without oxidation for 24 h. LDL and oxLDL treatments (≤4 μg) did not affect endothelial function as measured by permeability to FITC–dextran or cell viability (Supplementary Figure S1). To further explore whether the loss of endothelial function after 8 μg of oxLDL is due to protein or lipid fractions, LDL was sub-fractionated. LDL-L and oxLDL-L increased barrier permeability significantly in a concentration- dependent manner (Figure 1A), whereas LDL-protein had no significant effect (Supplementary Figure S2). Lipids fractionated from oxLDL and LDL caused a significant inhibition of metabolic activity after addition of >4 μg and 8 μg respectively (Figure 1B). Despite evidence of cell stress, measured as metabolic inhibition, there was no significant release of lactate dehydrogenase (LDH) when treated with 4 μg of LDL- or oxLDL-L (Supplementary Figure S3). Addition of LDL or lipids isolated from 4 μg of LDL led to a 3-fold increase in cellular cholesterol concentration (Supplementary Figure S4).

The effect of LDL and oxLDL-L on endothelial barrier permeability

Figure 1
The effect of LDL and oxLDL-L on endothelial barrier permeability

HMVEC (1×105cells) were seeded in Transwell inserts for 2 weeks before lipid treatments. HMVECs were treated with increasing concentrations of LDL-L and oxLDL-L for 24 h. Barrier tightness was measured by FITC–dextran permeability (A). Cell metabolic activity was measured by CellTiter-Blue® assay (B). The tight junction protein ZO-1 is stained green and nuclei stained blue with DAPI stain in HMVECs before (C) and after (D) treating with LDL-L (from 4 μg of LDL). **P<0.01, ***P<0.001, n=3 independent experiments.

Figure 1
The effect of LDL and oxLDL-L on endothelial barrier permeability

HMVEC (1×105cells) were seeded in Transwell inserts for 2 weeks before lipid treatments. HMVECs were treated with increasing concentrations of LDL-L and oxLDL-L for 24 h. Barrier tightness was measured by FITC–dextran permeability (A). Cell metabolic activity was measured by CellTiter-Blue® assay (B). The tight junction protein ZO-1 is stained green and nuclei stained blue with DAPI stain in HMVECs before (C) and after (D) treating with LDL-L (from 4 μg of LDL). **P<0.01, ***P<0.001, n=3 independent experiments.

To determine whether the disruption of tight-junction-associated protein was linked to loss of barrier integrity, we immunostained HMVEC for ZO-1 before and after LDL-lipid treatments. After treatment with a non-toxic but barrier-disturbing concentration of LDL-L (4 μg), ZO-1 staining was diffuse (Figure 1D) and ZO-1 was not visible on the margins of the cytoplasmic membrane surface; nevertheless, membranes appeared intact. Staining after LDL-L addition was unlike the ZO-1 staining pattern that was observed for untreated control HMVECs.

LDL- and oxLDL-lipids increase NF-κB-dependent basolateral secretion of TNF-α and IL-6

To explore whether LDL, oxLDL and sub fractions of LDL can trigger endothelial inflammatory responses, HMVECs were cultured on Transwells until a tight barrier was formed, then exposed apically to LDL- and oxLDL-L. Directional secretion of pro-inflammatory mediators was analysed in both apical and basolateral compartments. oxLDL-L significantly increased both apical and basolateral secretion of TNF-α (Figure 2A) and IL-6 (Figure 2B) compared with LDL-L-treated cells.

The effect of LDL-L and oxLDL-L on endothelial barrier inflammation

Figure 2
The effect of LDL-L and oxLDL-L on endothelial barrier inflammation

HMVECs (1×105 cells) were seeded in Transwell inserts for 2 weeks before lipid treatments. HMVECs were treated with increasing concentrations of LDL-L for 24 h (A and B) or LDL (4 μg; C and D). Cells were co-incubated with 20 μM SN50 before measuring IL-6 (A and C) and TNF-α (B and D) in both apical and basolateral media by ELISA. The level of cellular GSH (E) was determined by the GSH–Glo assay. Data are expressed as means ± S.E.M., *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n=3 independent experiments.

Figure 2
The effect of LDL-L and oxLDL-L on endothelial barrier inflammation

HMVECs (1×105 cells) were seeded in Transwell inserts for 2 weeks before lipid treatments. HMVECs were treated with increasing concentrations of LDL-L for 24 h (A and B) or LDL (4 μg; C and D). Cells were co-incubated with 20 μM SN50 before measuring IL-6 (A and C) and TNF-α (B and D) in both apical and basolateral media by ELISA. The level of cellular GSH (E) was determined by the GSH–Glo assay. Data are expressed as means ± S.E.M., *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n=3 independent experiments.

To determine whether the increase in cytokine secretion was due to activation of NF-KB (nuclear factor kappa-light-chain-enhancer of activated B cells), the inhibitory peptide SN50 was included when HMVECs were exposed to lipid fractions of LDL and oxLDL. SN50 significantly inhibited IL-6 (Figure 2C) and TNF-α (Figure 2D) production, without interfering with cell viability (Supplementary Figure S5), confirming the importance of NF-KB activation on the effects of LDL and oxLDL-L on HMVECs.

To understand whether the increase in cytokine secretion that is elicited by LDL-L and oxLDL-L in an NF-κB-dependent manner is associated with an altered redox state, we examined intracellular GSH in HMVECs after over 2 h of lipid treatments. LDL-L (oxidized and control) significantly depleted cellular GSH concentration (Figure 2E) and this mirrored the patterns of cytokine secretion.

Effect of hypercholesterolaemia and statin intervention on LDL oxidation in mid-life

Statin intervention in mid-life is reported to reduce incidence of dementia in later life, therefore we have undertaken a qualitative and quantitative analysis of lipid oxidation products in LDL from hypercholesterolaemic patients compared with age-matched controls whose cholesterol levels are within the normal range; their baseline characteristics are reported in Table 1. 

Table 1
Demographics of healthy control and hypercholesterolaemic patient populations

The healthy control subjects continued a normal lifestyle and hypercholesterolaemic patients received simvastatin (40 mg/day) for 3 months.

 Baseline Three months follow-up 
 Control (n=10) Hyper-cholesterolaemic (n=10) P Control (n=10) Hyper-cholesterolaemic (n=10) P 
Weight (kg) 62±2.47 63.8±2.69 >0.05 61±2.3 64±2.7 >0.05 
Body mass index (BMI; kg/m224.88±0.74 26.35±1.1 >0.05 24.7±0.68 26.3±1.2 >0.05 
Cholesterol (mM) 4.08±0.18 6.72±0.78 <0.001 3.8±0.13 4.63±0.31 >0.05 
Age (years) 46.4±1.7 47.4±1.7 >0.05 46.4±1.7 47.4±1.7 >0.05 
 Baseline Three months follow-up 
 Control (n=10) Hyper-cholesterolaemic (n=10) P Control (n=10) Hyper-cholesterolaemic (n=10) P 
Weight (kg) 62±2.47 63.8±2.69 >0.05 61±2.3 64±2.7 >0.05 
Body mass index (BMI; kg/m224.88±0.74 26.35±1.1 >0.05 24.7±0.68 26.3±1.2 >0.05 
Cholesterol (mM) 4.08±0.18 6.72±0.78 <0.001 3.8±0.13 4.63±0.31 >0.05 
Age (years) 46.4±1.7 47.4±1.7 >0.05 46.4±1.7 47.4±1.7 >0.05 

LDL isolated from statin-naive hypercholesterolaemic subjects showed a trend for higher electrophoretic mobility at baseline by agarose gel electrophoresis (RF=0.53±0.06) compared with control subjects (RF=0.46±0.05; Figure 3A). To investigate levels of circulating protein and lipid oxidation in the patients and controls, plasma was analysed for protein carbonyls and 8-isoprostane F2α (Figures 3B and 3C). Statin-naive hyperlipidaemic subjects had higher plasma carbonyls (3.95±0.2 nmol/mg; P<0.001) and 8-isoprostane F2α (43.5±8.42 pg/ml; P<0.01) compared with control subjects (2.65±0.15 nmol/mg and 24.2±5.37 pg/ml respectively). Statin treatment for 3 months significantly decreased blood cholesterol levels (from 6.72±0.78 mM to 4.63±0.31 mM; P<0.001), plasma carbonyl (2.5±0.4; P<0.001) and 8-isoprostane F2α levels (30.4±4.0 nmol/mg; P<0.05).

LDL from statin-naive hypercholesterolaemic patients is more oxidized than LDL from healthy controls and statin treatments reduced LDL oxidation

Figure 3
LDL from statin-naive hypercholesterolaemic patients is more oxidized than LDL from healthy controls and statin treatments reduced LDL oxidation

Following density gradient ultracentrifugation, LDL electronegativity was examined by agarose gel electrophoresis (A). Protein carbonyls (B) and 8-isoprostane F2α (C) were determined in plasma from ten controls and ten patients analysed in triplicate by ELISA. **P<0.01 and ***P<0.001.

Figure 3
LDL from statin-naive hypercholesterolaemic patients is more oxidized than LDL from healthy controls and statin treatments reduced LDL oxidation

Following density gradient ultracentrifugation, LDL electronegativity was examined by agarose gel electrophoresis (A). Protein carbonyls (B) and 8-isoprostane F2α (C) were determined in plasma from ten controls and ten patients analysed in triplicate by ELISA. **P<0.01 and ***P<0.001.

Effect of LDL from hypercholesterolaemic patients pre- and post-statin intervention on endothelial function

Compared with HMVEC treatment with the LDL-L (4 μg of LDL) from normolipidaemic subjects, LDL-L from hyperlipidaemic subjects increased endothelial permeability as observed by FITC–dextran permeability (Figure 4A; P<0.05) and immunohistochemistry for tight junctions was decreased (Figures 4B and 4C; P<0.001). At the baseline, the percentage of endothelial cells with tight junctions was significantly decreased upon patient LDL-L treatment. After 3 months of statin intervention, LDL-L did not change the percentage of cells expressing junctional ZO-1 compared with control LDL-L. At the baseline, LDL-L from patients decreased GSH (15.94±1.0 nmol/mg compared with 12.2±0.6 nmol/mg; P<0.01; untreated cells 22.75±1.2 nmol/mg of protein; n=10/group; Figure 4D). After 3 months of treatment with statins, there was no difference between the effects of LDL isolated from either patients or controls on HMVEC function.

Inflammatory effects of LDL-L are removed by statin intervention in hypercholesterolaemic patients

Figure 4
Inflammatory effects of LDL-L are removed by statin intervention in hypercholesterolaemic patients

LDL-L (4 μg) from normolipidaemic (n=10) and statin-naive hypercholesterolaemic subject plasma (n=10) pre- and post-statin intervention were investigated for effects on HMVEC barrier function in duplicate (A); distribution of tight junction protein ZO-1 in duplicate (B and C); intracellular GSH concentration by GSH–Glo in duplicate (D); secreted IL-6 by ELISA in duplicate (E); and TNF-α by ELISA in duplicate (F). *P<0.05, **P<0.01, ***P<0.001.

Figure 4
Inflammatory effects of LDL-L are removed by statin intervention in hypercholesterolaemic patients

LDL-L (4 μg) from normolipidaemic (n=10) and statin-naive hypercholesterolaemic subject plasma (n=10) pre- and post-statin intervention were investigated for effects on HMVEC barrier function in duplicate (A); distribution of tight junction protein ZO-1 in duplicate (B and C); intracellular GSH concentration by GSH–Glo in duplicate (D); secreted IL-6 by ELISA in duplicate (E); and TNF-α by ELISA in duplicate (F). *P<0.05, **P<0.01, ***P<0.001.

LDL-lipids from patients with Alzheimer's disease act as inflammatory triggers in microvascular endothelial cells

To explore whether LDL-L from patients with dementia are more oxidized than controls and to investigate their inflammatory effects, the endothelial model was treated with 4 μg of LDL-L from each patient individually from the three groups, AD, AD-plus and control subjects. Although the total cholesterol and LDL cholesterol levels between subject groups were not different (Table 2), AD-plus patients had higher plasma 8-isoprostane F2α levels (Figure 5A; 39.93±3 pg/ml) compared with the healthy control group (30.08±1.8 pg/ml). There was an increase in endothelial permeability with AD and AD-plus LDL-L treatments and intracellular GSH levels were decreased (23.8±1.2 compared with 16.5±0.7 nmol/mg of protein). AD-plus LDL-L were also more inflammatory as measured by basolateral secretion of IL-6 (80.8±1 compared with 18.6±3.6 pg/ml).

Table 2
Demographics of mid-life adult volunteers
 Control (n=10) AD (n=10) AD-plus (n=10) P 
Age (years) 73±2.5 81±1.4 80±1.7 >0.05 
Body mass index (BMI; kg/m223.7±0.34 24.1±0.69 25.8±0.36 >0.05 
Total cholesterol (mmol/l) 5.67±0.17 5.24±0.32 4.9±0.2 >0.05 
LDL cholesterol (mg/dl) 121.5±7.6 113.2±11.1 108.3±10.0 >0.05 
 Control (n=10) AD (n=10) AD-plus (n=10) P 
Age (years) 73±2.5 81±1.4 80±1.7 >0.05 
Body mass index (BMI; kg/m223.7±0.34 24.1±0.69 25.8±0.36 >0.05 
Total cholesterol (mmol/l) 5.67±0.17 5.24±0.32 4.9±0.2 >0.05 
LDL cholesterol (mg/dl) 121.5±7.6 113.2±11.1 108.3±10.0 >0.05 

LDL-L are more oxidized and inflammatory in AD (n=10) and AD-plus patients (n=10) compared with controls (n=10)

Figure 5
LDL-L are more oxidized and inflammatory in AD (n=10) and AD-plus patients (n=10) compared with controls (n=10)

Plasma 8-isoprostane F2α was determined for each plasma in triplicate (A). LDL-L (4 μg) from patients with AD and AD-plus were investigated for effects on microvascular endothelial barrier function (B) and intracellular GSH concentration by GSH–Glo assay (C) in duplicate for each plasma. Secreted IL-6 (D) levels were measured by IL-6 ELISA in duplicate for each plasma. NS, not significant. *P<0.05, **P<0.01 and ***P<0.001.

Figure 5
LDL-L are more oxidized and inflammatory in AD (n=10) and AD-plus patients (n=10) compared with controls (n=10)

Plasma 8-isoprostane F2α was determined for each plasma in triplicate (A). LDL-L (4 μg) from patients with AD and AD-plus were investigated for effects on microvascular endothelial barrier function (B) and intracellular GSH concentration by GSH–Glo assay (C) in duplicate for each plasma. Secreted IL-6 (D) levels were measured by IL-6 ELISA in duplicate for each plasma. NS, not significant. *P<0.05, **P<0.01 and ***P<0.001.

DISCUSSION

In the present study, we show that the activation of microvascular endothelial cells with LDL-L increased the secretion of the inflammatory mediators TNF-α and IL-6 and decreased the membrane localization of the tight junction protein, ZO-1. LDL-L isolated from patients with hypercholesterolaemia were more oxidized and inflammatory towards endothelial cells than those from healthy age-matched subjects. Similarly, LDL-L from AD-plus patients were found to contain a higher level of lipid peroxidation and promoted more inflammatory IL-6 secretion from endothelial cells compared with controls. After, simvastatin intervention for 3 months in hypercholesterolaemic patients, LDL-L oxidation was decreased and the inflammatory effects of oxLDL on endothelial cells were reduced. These findings support the hypothesis that elevated cholesterol in mid-life increases the concentrations of oxidized LDL molecules which in turn can elicit a directional inflammatory response and results in tissue inflammation. It has been described previously that inflammatory cytokines secreted by endothelial cells can exert an autocrine disruption of endothelial tight junction integrity [23]. During neurodegenerative conditions, the barrier property of brain microvascular endothelial cells is thought to be damaged by disruption of junctional proteins resulting in barrier leakage [24]. Moreover, cerebral endothelium disruption and white matter lesions typical of dementia are observed more frequently in diabetes and hypercholesterolaemic patients compared with healthy controls [25].

The BBB consists of microvascular endothelial cells that are adjoined by specific protein tight junctions (e.g. occludins, ZO-1, ZO-2, ZO-3, claudins and cingulins) with their basement membrane underlying brain pericytes and astroglial foot processes [25]. In healthy brains these layers display specific and tightly regulated transport mechanisms between the blood and the ventricular cerebrospinal fluid [25]. Although normal aging increases BBB permeability [25], oxLDL promotes endothelial activation including increased monocyte adhesion to the vascular wall and induction of NF-κB. There is controversy over whether aging alone associates with increased BBB permeability; Pelegri et al. [26] have reported an early increase in permeability in the senescence-accelerated mouse model Senescence Accelerated Mouse Phenotype (SAMP)-8 at 12 months which is not present in control animals and is suggested to underlie deficits in learning and memory. SAMP-8 mice are also characterized by early loss of antioxidant enzymes, i.e. with altered control of the scavenging of oxidative species [27].

In a model of localized excitotoxicity-induced neurodegeneration, BBB breakdown in itself was not sufficient to elicit cell death; a subsequent peroxynitrite-mediated event was required [28]. Peroxynitrite is lipophilic and can cross membranes via diffusion in the absence of a transporter. Although the BBB requires the presence of carrier/proteins and transporters for rapid molecular transport of nutrients, e.g. fatty acids, direct uptake of lipid hydroperoxides proceeds at an 8-fold higher rate than diffusion-controlled uptake of parent lipids [29]. Thus, clear pathways exist for transport of reactive species and modified lipids from the periphery to the CNS where they are neurotoxic to hippocampal neurones.

To better understand whether oxLDL was disruptive to an endothelial barrier, in vitro minimally modified LDL was prepared [19] from a pool of healthy subjects. This approach was taken to represent range of oxidation present in LDL. A CuSO4-based method to prepare minimally oxLDL is well characterized in the literature showing similar levels of oxidation to those seen in patients with vascular disease [24]. In line with the literature, oxLDL displayed significantly higher inflammatory activity towards endothelial cells than freshly prepared LDL from control subjects [30]. Prior to ascribing physiological significance to these findings, oxLDL lipid effects on the BBB should be tested in vivo.

After the LDL was subfractionated, it was found to be the lipid but not the protein components that mediated cell stress. We did not measure any quantitative difference in the uptake of lipids whether they were delivered by LDL or bound to albumin after 24 h. This does not preclude a possible difference in rate of delivery. oxLDL-L elicited the greatest loss of GSH which has been reported by others to lead to an increase in NF-κB activation in endothelial cells [31]. In turn, NF-κB can regulate multiple pro-inflammatory target genes [21]. NF-κB activation and downstream expression of cytokines is controlled at least in part by the intracellular redox state. A shift towards a more oxidized environment increases phosphatase oxidation and inactivation, thus propagating inhibitor of NF-κB kinase (IKK) cascade activity and IKK dissociation from NF-κB [23].

oxLDL are known to cause barrier disruption in mice which has been attributed to a decrease in the expression of actin-depolymerizing factor (ADF) [32]. Others showed that overexpression of ADF attenuated oxLDL-induced disruption of endothelial barrier marked by restoration of trans-endothelial electrical resistance, permeability of Evans Blue and expression of tight-junction-associated proteins including ZO-1 and occludin and blocked oxLDL-induced oxidative stress [22]. Differential expression of inflammatory and actin-remodelling proteins by endothelial cells has also been shown after exposure to serum from patients with chronic kidney disease [33] suggesting a common association between endothelial cell response to plasma components.

Consistent with previous reports, we observed that LDL is more oxidized in hypercholesterolaemic patients [17]. Of note, plasma protein carbonyl and isoprostane concentrations described previously in AD-plus patients [14,20] were not different from those reported in the present study for hypercholesterolaemic patients. Importantly, after patients were treated with statins, their LDL oxidation measured as both protein and lipid oxidation was no different from that of healthy control subjects. However, we acknowledge the fact that the present study involved a small cohort size (n=20) and this limits the statistical power.

The present data show for the first time that lipids from LDL of untreated hypercholesterolaemic patients trigger the loss of microvascular barrier integrity. Moreover, the increased lipid toxicity in LDL from hypercholesterolaemic patients compared with control subject LDL towards microvascular endothelial cells was prevented after intervention with simvastatin for 3 months. In a similar manner, when LDL was examined from patients with AD and AD-plus, isoprostane concentrations were higher as previously described [14]. Moreover, their lipids were more pro-inflammatory to endothelial cells promoting a significant increase in basolateral IL-6 directional secretion associated with loss of GSH. When considered alongside the importance of BBB integrity for brain health, these observations suggest an explanation for the observed reduction in dementia incidence for those receiving statins, independently of a reduction in plasma cholesterol and which may be attributed to statin effects on lipid oxidation [34].

These data support the hypothesis that statins exert a protective effect against microvascular damage, modulating LDL by a mechanism that may not only be dependent on reducing cholesterol concentration but may be due to their capacity to prevent LDL oxidation. This may account for the protective effects of statin usage in mid-life against later dementia development and offers a rationale for managing patients with elevated oxLDL and mild or subjective cognitive impairment using statins.

In conclusion, the present study demonstrated that LDL-L from AD and AD-plus patients are more damaging to endothelial barrier properties and increase release of inflammatory cytokines. These data support the hypothesis that in vivo intervention with statins modifies LDL-L oxidation and exerts a protective effect against microvascular damage in a manner that is independent of cholesterol concentration. This may account for the protective effects of statin usage in mid-life against the later development of AD.

AUTHOR CONTRIBUTION

H. Irundika Dias collected samples from hypercholesterolaemic subjects, undertook experiments, data analysis and manuscript preparation. Caroline Brown undertook cell culture experiments, viability and cytokine analyses. Gregory Lip supervised patient recruitment, data analysis and manuscript preparation. M. Cristina Polidori collected samples from AD and AD-plus patient groups, data analysis and manuscript preparation. Helen Griffiths designed the study, analysed data and drafted the manuscript. All authors read and approved the final manuscript.

We acknowledge technical support of Ms Charlotte Bland, Aston Research Centre for Healthy Ageing, on the confocal scanning multiphoton microscope.

FUNDING

This work was supported by the Dunhill Medical Trust [grant number R92/1108].

Abbreviations

     
  • AD

    Alzheimer's disease

  •  
  • ADF

    actin-depolymerizing factor

  •  
  • AD-plus

    AD with vascular dementia

  •  
  • BBB

    blood–brain barrier

  •  
  • CNS

    central nervous system

  •  
  • GSH

    glutathione

  •  
  • HMVEC

    human microvascular endothelial cell

  •  
  • IKK

    inhibitor of NF-κB kinase; IL, interleukin

  •  
  • LDL

    low-density lipoprotein

  •  
  • LDL-L

    LDL-lipids

  •  
  • NF-kB

    nuclear factor kappa-light-chain-enhancer of activated B cells

  •  
  • oxLDL

    oxidized LDL

  •  
  • oxLDL-L

    oxLDL-lipids

  •  
  • TNF

    tumour necrosis factor

  •  
  • ZO-1

    zonula occludens-1

References

References
1
Vance
J.E.
Hayashi
H.
Karten
B.
Cholesterol homeostasis in neurons and glial cells
Semin. Cell Dev. Biol.
2005
, vol. 
16
 (pg. 
193
-
212
)
[PubMed]
2
Abbott
N.J.
Ronnback
L.
Hansson
E.
Astrocyte-endothelial interactions at the blood-brain barrier
Nat. Rev. Neurosci.
2006
, vol. 
7
 (pg. 
41
-
53
)
[PubMed]
3
Abbott
N.J.
Patabendige
A.A.K.
Dolman
D.E.M.
Yusof
S.R.
Begley
D.J.
Structure and function of the blood-brain barrier
Neurobiol. Dis.
2010
, vol. 
37
 (pg. 
13
-
25
)
[PubMed]
4
Turley
S.D.
Burns
D.K.
Rosenfeld
C.R.
Dietschy
J.M.
Brain does not utilize low density lipoprotein-cholesterol during fetal and neonatal development in the sheep
J. Lipid. Res
1996
, vol. 
37
 (pg. 
1953
-
1961
)
[PubMed]
5
Dietschy
J.M.
Kita
T.
Suckling
K.E.
Goldstein
J.L.
Brown
M.S.
Cholesterol synthesis in vivo and in vitro in the WHHL rabbit, an animal with defective low density lipoprotein receptors
J. Lipid Res.
1983
, vol. 
24
 (pg. 
469
-
480
)
[PubMed]
6
Yu
L.
von Bergmann
K.
Lutjohann
D.
Hobbs
H.H.
Cohen
J.C.
Selective sterol accumulation in ABCG5/ABCG8-deficient mice
J. Lipid Res.
2004
, vol. 
45
 (pg. 
301
-
307
)
[PubMed]
7
Cramer
C.
Haan
M.N.
Galea
S.
Langa
K.M.
Kalbfleisch
J.D.
Use of statins and incidence of dementia and cognitive impairment without dementia in a cohort study
Neurology
2008
, vol. 
71
 (pg. 
344
-
350
)
[PubMed]
8
Satoh
M.
Takahashi
Y.
Tabuchi
T.
Minami
Y.
Tamada
M.
Takahashi
K.
Itoh
T.
Morino
Y.
Nakamura
M.
Cellular and molecular mechanisms of statins: an update on pleiotropic effects
Clin. Sci.
2015
, vol. 
129
 (pg. 
93
-
105
)
[PubMed]
9
Lahera
V.
Goicoechea
M.
de Vinuesa
S.G.
Miana
M.
de las Heras
N.
Cachofeiro
V.
Luño
J.
Endothelial dysfunction, oxidative stress and inflammation in atherosclerosis: beneficial effects of statins
Curr. Med. Chem.
2007
, vol. 
14
 (pg. 
243
-
248
)
[PubMed]
10
Li
G.
Larson
E.B.
Sonnen
J.A.
Shofer
J.B.
Petrie
E.C.
Schantz
A.
Peskind
E.R.
Raskind
M.A.
Breitner
J.C.
Montine
T.J.
Statin therapy is associated with reduced neuropathologic changes of Alzheimer disease
Neurology
2007
, vol. 
69
 (pg. 
878
-
885
)
[PubMed]
11
Li
G.
Shofer
J.B.
Rhew
I.C.
Kukull
W.A.
Peskind
E.R.
McCormick
W.
Bowen
J.D.
Schellenberg
G.D.
Crane
P.K.
Breitner
J.C.
Larson
E.B.
Age-varying association between statin use and incident Alzheimer's disease
J. Am. Geriatr. Soc.
2010
, vol. 
58
 (pg. 
1311
-
1317
)
[PubMed]
12
Chauhan
N.B.
Siegel
G.J.
Feinstein
D.L.
Effects of lovastatin and pravastatin on amyloid processing and inflammatory response in TgCRND8 brain
Neurochem. Res.
2004
, vol. 
29
 (pg. 
1897
-
1911
)
[PubMed]
13
Hjuler Nielsen
M.
Irvine
H.
Vedel
S.
Raungaard
B.
Beck-Nielsen
H.
Handberg
A.
Elevated atherosclerosis-related gene expression, monocyte activation and microparticle-release are related to increased lipoprotein-associated oxidative stress in familial hypercholesterolemia
PLoS One
2015
, vol. 
10
 pg. 
e0121516
 
[PubMed]
14
Polidori
M.C.
Mattioli
P.
Aldred
S.
Cecchetti
R.
Stahl
W.
Griffiths
H.
Senin
U.
Sies
H.
Mecocci
P.
Plasma antioxidant status, immunoglobulin g oxidation and lipid peroxidation in demented patients: relevance to Alzheimer disease and vascular dementia
Dement. Geriatr. Cogn. Disord.
2004
, vol. 
18
 (pg. 
265
-
270
)
[PubMed]
15
Griffiths
H.R.
Aldred
S.
Dale
C.
Nakano
E.
Kitas
G.D.
Grant
M.G.
Nugent
D.
Taiwo
F.A.
Li
L.
Powers
H.J.
Homocysteine from endothelial cells promotes LDL nitration and scavenger receptor uptake
Free Radic. Biol. Med.
2006
, vol. 
40
 (pg. 
488
-
500
)
[PubMed]
16
Pratico
D.
Clark
C.M.
Liun
F.
Rokach
J.
Lee
V.Y.
Trojanowski
J.Q.
Increase of brain oxidative stress in mild cognitive impairment: a possible predictor of Alzheimer disease
Arch. Neurol.
2002
, vol. 
59
 (pg. 
972
-
976
)
[PubMed]
17
Ishigaki
Y.
Katagiri
H.
Gao
J.
Yamada
T.
Imai
J.
Uno
K.
Hasegawa
Y.
Kaneko
K.
Ogihara
T.
Ishihara
H.
, et al. 
Impact of plasma oxidized low-density lipoprotein removal on atherosclerosis
Circulation
2008
, vol. 
118
 (pg. 
75
-
83
)
[PubMed]
18
Jick
H.
Zornberg
G.L.
Jick
S.S.
Seshadri
S.
Drachman
D.A.
Statins and the risk of dementia
Lancet
2000
, vol. 
356
 (pg. 
1627
-
1631
)
[PubMed]
19
Li
L.
Willets
R.S.
Polidori
M.C.
Stahl
W.
Nelles
G.
Sies
H.
Griffiths
H.R.
Oxidative LDL modification is increased in vascular dementia and is inversely associated with cognitive performance
Free Radic. Res
2010
, vol. 
44
 (pg. 
241
-
248
)
[PubMed]
20
Dias
I.H.
Mistry
J.
Fell
S.
Reis
A.
Spickett
C.M.
Polidori
M.C.
Lip
G.Y.H.
Griffiths
H.R.
Oxidized LDL lipids increase β-amyloid production by SH-SY5Y cells through glutathione depletion and lipid raft formation
Free Radic. Biol. Med.
2014
, vol. 
75
 (pg. 
48
-
59
)
[PubMed]
21
Stewart
J.C.
Colorimetric determination of phospholipids with ammonium ferrothiocyanate
Anal. Biochem.
1980
, vol. 
104
 (pg. 
10
-
14
)
[PubMed]
22
Griffiths
H.R.
Aldred
S.
Dale
C.
Nakano
E.
Kitas
G.D.
Grant
M.G.
Nugent
D.
Taiwo
F.A.
Li
L.
Powers
H.J.
Homocysteine from endothelial cells promotes LDL nitration and scavenger receptor uptake
Free Radic. Biol. Med.
2006
, vol. 
40
 (pg. 
488
-
500
)
[PubMed]
23
Lutgendorf
M.A.
Ippolito
D.L.
Mesngon
M.T.
Tinnemore
D.
Dehart
M.J.
Dolinsky
B.M.
Napolitano
P.G.
Effect of dexamethasone administered with magnesium sulfate on inflammation-mediated degradation of the blood–brain barrier using an in vitro model
Reprod. Sci.
2014
, vol. 
21
 (pg. 
483
-
491
)
[PubMed]
24
Ehara
S.
Ueda
M.
Naruko
T.
Haze
K.
Itoh
A.
Otsuka
M.
Komatsu
R.
Matsuo
T.
Itabe
H.
Takano
T.
, et al. 
Elevated levels of oxidized low density lipoprotein show a positive relationship with the severity of acute coronary syndromes
Circulation
2001
, vol. 
103
 (pg. 
1955
-
1960
)
[PubMed]
25
Chui
H.
Ramirez-Gomez
L.
Clinical and imaging features of mixed Alzheimer and vascular pathologies
Alzheimers Res. Ther.
2015
, vol. 
7
 pg. 
21
 
[PubMed]
26
Pelegri
C.
Canudas
A.M.
del Valle
J.
Casadesus
G.
Smith
M.A.
Camins
A.
Pallas
M.
Vilaplana
J.
Increased permeability of blood-brain barrier on the hippocampus of a murine model of senescence
Mech. Ageing Dev.
2007
, vol. 
128
 (pg. 
522
-
528
)
[PubMed]
27
Sureda
F.X.
Gutierrez-Cuesta
J.
Romeu
M.
Mulero
M.
Canudas
A.M.
Camins
A.
Mallol
J.
Pallas
M.
Changes in oxidative stress parameters and neurodegeneration markers in the brain of the senescence-accelerated mice SAMP-8
Exp. Gerontol.
2006
, vol. 
41
 (pg. 
360
-
367
)
[PubMed]
28
Parathath
S.R.
Parathath
S.
Tsirka
S.E.
Nitric oxide mediates neurodegeneration and breakdown of the blood-brain barrier in tPA-dependent excitotoxic injury in mice
J. Cell Sci.
2006
, vol. 
119
 (pg. 
339
-
349
)
[PubMed]
29
Vila
A.
Levchenko
V.V.
Korytowski
W.
Girotti
A.W.
Sterol carrier protein-2-facilitated intermembrane transfer of cholesterol- and phospholipid-derived hydroperoxides
Biochemistry
2004
, vol. 
43
 (pg. 
12592
-
12605
)
[PubMed]
30
Saing
L.
Wei
Y.-C.
Tseng
C.-J.
Ergothioneine represses inflammation and dysfunction in human endothelial cells exposed to oxidized low-density lipoprotein
Clin. Exp. Pharmacol. Physiol.
2015
[PubMed]
31
Zeng
Q.
Song
R.
Ao
L.
Xu
D.
Venardos
N.
Fullerton
D.A.
Meng
X.
Augmented osteogenic responses in human aortic valve cells exposed to oxLDL and TLR4 agonist: a mechanistic role of Notch1 and NF-κB interaction
PLoS One
2014
, vol. 
9
 pg. 
e95400
 
[PubMed]
32
Wang
J.
Sun
L.
Si
Y.F.
Li
B.M.
Overexpression of actin-depolymerizing factor blocks oxidized low-density lipoprotein-induced mouse brain microvascular endothelial cell barrier dysfunction
Mol. Cell. Biochem.
2012
, vol. 
371
 (pg. 
1
-
8
)
[PubMed]
33
Carbó
C.
Arderiu
G.
Escolar
G.
Fusté
B.
Cases
A.
Carrascal
M.
Abián
J.
Díaz-Ricart
M.
Differential expression of proteins from cultured endothelial cells exposed to uremic versus normal serum
J. Kidney Dis.
2008
, vol. 
51
 (pg. 
603
-
612
)
[PubMed]
34
Yoshida
H.
Shoda
T.
Yanai
H.
Ikewaki
K.
Kurata
H.
Ito
K.
Furutani
N.
Tada
N.
Witztum
J.L.
Tsimikas
S.
Effects of pitavastatin and atorvastatin on lipoprotein oxidation biomarkers in patients with dyslipidemia
Atherosclerosis
2013
, vol. 
226
 (pg. 
161
-
164
)
[PubMed]

Supplementary data