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1.
Role of Phagocytosis in the Pro-Inflammatory Response in LDL-Induced Foam Cell Formation; a Transcriptome Analysis.
Orekhov, AN, Nikiforov, NG, Sukhorukov, VN, Kubekina, MV, Sobenin, IA, Wu, WK, Foxx, KK, Pintus, S, Stegmaier, P, Stelmashenko, D, et al
International journal of molecular sciences. 2020;(3)
Abstract
Excessive accumulation of lipid inclusions in the arterial wall cells (foam cell formation) caused by modified low-density lipoprotein (LDL) is the earliest and most noticeable manifestation of atherosclerosis. The mechanisms of foam cell formation are not fully understood and can involve altered lipid uptake, impaired lipid metabolism, or both. Recently, we have identified the top 10 master regulators that were involved in the accumulation of cholesterol in cultured macrophages induced by the incubation with modified LDL. It was found that most of the identified master regulators were related to the regulation of the inflammatory immune response, but not to lipid metabolism. A possible explanation for this unexpected result is a stimulation of the phagocytic activity of macrophages by modified LDL particle associates that have a relatively large size. In the current study, we investigated gene regulation in macrophages using transcriptome analysis to test the hypothesis that the primary event occurring upon the interaction of modified LDL and macrophages is the stimulation of phagocytosis, which subsequently triggers the pro-inflammatory immune response. We identified genes that were up- or downregulated following the exposure of cultured cells to modified LDL or latex beads (inert phagocytosis stimulators). Most of the identified master regulators were involved in the innate immune response, and some of them were encoding major pro-inflammatory proteins. The obtained results indicated that pro-inflammatory response to phagocytosis stimulation precedes the accumulation of intracellular lipids and possibly contributes to the formation of foam cells. In this way, the currently recognized hypothesis that the accumulation of lipids triggers the pro-inflammatory response was not confirmed. Comparative analysis of master regulators revealed similarities in the genetic regulation of the interaction of macrophages with naturally occurring LDL and desialylated LDL. Oxidized and desialylated LDL affected a different spectrum of genes than naturally occurring LDL. These observations suggest that desialylation is the most important modification of LDL occurring in vivo. Thus, modified LDL caused the gene regulation characteristic of the stimulation of phagocytosis. Additionally, the knock-down effect of five master regulators, such as IL15, EIF2AK3, F2RL1, TSPYL2, and ANXA1, on intracellular lipid accumulation was tested. We knocked down these genes in primary macrophages derived from human monocytes. The addition of atherogenic naturally occurring LDL caused a significant accumulation of cholesterol in the control cells. The knock-down of the EIF2AK3 and IL15 genes completely prevented cholesterol accumulation in cultured macrophages. The knock-down of the ANXA1 gene caused a further decrease in cholesterol content in cultured macrophages. At the same time, knock-down of F2RL1 and TSPYL2 did not cause an effect. The results obtained allowed us to explain in which way the inflammatory response and the accumulation of cholesterol are related confirming our hypothesis of atherogenesis development based on the following viewpoints: LDL particles undergo atherogenic modifications that, in turn, accompanied by the formation of self-associates; large LDL associates stimulate phagocytosis; as a result of phagocytosis stimulation, pro-inflammatory molecules are secreted; these molecules cause or at least contribute to the accumulation of intracellular cholesterol. Therefore, it became obvious that the primary event in this sequence is not the accumulation of cholesterol but an inflammatory response.
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Immune Complexes and the Risk of CVD in Type 1 Diabetes.
Lopes-Virella, MF, Bebu, I, Hunt, KJ, Virella, G, Baker, NL, Braffett, B, Gao, X, Lachin, JM, ,
Diabetes. 2019;(9):1853-1860
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Abstract
We investigated whether the composition of modified forms of LDL in circulating immune complexes (LDL-ICs) was associated with cardiovascular disease (CVD) outcomes, including any CVD, major adverse cardiac and cerebrovascular events (MACCE), myocardial infarction (MI), and coronary artery disease, in type 1 diabetes (T1D). Our results demonstrate that the baseline levels of oxidized LDL (oxLDL), MDA-modified LDL (MDA-LDL), and advanced glycosylation-modified LDL (AGE-LDL) in circulating ICs were associated with the four CVD outcomes in unadjusted models, and adjustment by age and mean HbA1c only resulted in minimal reduction of these associations. After adjustments were made for other cardiovascular risk factors, particularly LDL cholesterol, oxLDL-IC and MDA-LDL-IC remained independently associated with the risk of CVD, and oxLDL-IC was independently associated with the risk of MACCE and MI. In the majority of cases, the baseline levels of modified LDL-IC (measured many years before the occurrence of any CVD event) were associated with the risk of CVD over a 25-year period even after adjustment for other risk factors (including LDL cholesterol). Therefore, modified LDL biomarkers may help identify patients with T1D at high risk for MACCE and CVD events very early in the evolution of the disease, before other signals of disease are apparent.
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Mediterranean meal versus Western meal effects on postprandial ox-LDL, oxidative and inflammatory gene expression in healthy subjects: a randomized controlled trial for nutrigenomic approach in cardiometabolic risk.
De Lorenzo, A, Bernardini, S, Gualtieri, P, Cabibbo, A, Perrone, MA, Giambini, I, Di Renzo, L
Acta diabetologica. 2017;(2):141-149
Abstract
AIMS: Inflammation and oxidative damage contribute significantly to the development of cardiovascular diseases (CVD). Postprandial oxidative stress and inflammation are characterized by an increased susceptibility of the organism toward oxidative damage after consumption of a meal rich in lipids and/or carbohydrates. Micronutrients modulate immune system and exert a protective action by reducing oxidized low-density lipoprotein (ox-LDL) level. The aim of the present study was to evaluate the postprandial plasma ox-LDL level and the gene expression of 13 genes related to oxidative stress (HOSp) and human inflammasome pathways (HIp), after a tocopherol-enriched Mediterranean meal (TEM), and a Western high-fat meal (HFM). Moreover, Mediterranean Adequacy Index was calculated to define the quality of both meals. METHODS We set up a randomized and crossover trial in healthy human volunteers. Ox-LDL level was measured by enzyme-linked immunosorbent assay and the gene expression of 13 genes related to HOSp and HIp by qRT-PCR. RESULTS Ox-LDL levels significantly decreased comparing HFM versus TEM (p < 0.05). Percentages of significantly overexpressed genes after each dietary treatment are as follows: (A) baseline versus HFM: 7.69 % HIp and 23.08 % HOSp; (B) baseline versus TEM: 7.69 % HIp and 7.69 % HOSp; (C) HFM versus TEM: 15.38 % HIp and 15.38 % HOSp. CONCLUSIONS TEM reduced postprandial risk factors of CVD, such as ox-LDL, and the expression of inflammation and oxidative stress-related genes. Chronic studies on larger population are necessary before definitive conclusions. TRIAL REGISTRATION ClinicalTrials.gov Id: NCT01890070.
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Post-prandial effects of hazelnut-enriched high fat meal on LDL oxidative status, oxidative and inflammatory gene expression of healthy subjects: a randomized trial.
Di Renzo, L, Merra, G, Botta, R, Gualtieri, P, Manzo, A, Perrone, MA, Mazza, M, Cascapera, S, De Lorenzo, A
European review for medical and pharmacological sciences. 2017;(7):1610-1626
Abstract
OBJECTIVE Postprandial oxidative stress is characterized by an increased susceptibility of the organism towards oxidative damage after consumption of a meal rich in lipids and/or carbohydrates. Micronutrients modulate the immune system and exert a protective action by reducing low-density lipoproteins oxidation (ox-LDL) via induction of antioxidant enzymes. SUBJECTS AND METHODS The clinical study was a randomized and cross-over trial, conducted through the CONSORT flowchart. We evaluated the gene expression of 103 genes related to oxidative stress (HOSp) and human inflammasome pathways (HIp), and ox-LDL level at fasting and after 40 g raw "Tonda Gentile delle Langhe" hazelnut consumption, in association with a McDonald's® Meal (McDM) in 22 healthy human volunteers. RESULTS Ox-LDL levels significantly increased comparing no dietary treatment (NDT) vs. McDM, and decreased comparing McDM vs. McDM + H (p<0.05). Percentage of significant genes expressed after each dietary treatment were the follows: (A) NDT vs. McDM: 3.88% HIp and 17.48% HOSp; (B) NDT vs. McDM + H: 17.48% HIp and 23.30% HOSp; (C) McDM vs. McDM + H: 17.48% HIp and 33.98% HOSp. CONCLUSIONS Hazelnut consumption reduced post prandial risk factors of atherosclerosis, such as ox-LDL, and the expression of inflammation and oxidative stress related genes. Chronic studies on larger population are necessary before definitive conclusions.
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Early Transcriptomic Response to LDL and oxLDL in Human Vascular Smooth Muscle Cells.
Damián-Zamacona, S, Toledo-Ibelles, P, Ibarra-Abundis, MZ, Uribe-Figueroa, L, Hernández-Lemus, E, Macedo-Alcibia, KP, Delgado-Coello, B, Mas-Oliva, J, Reyes-Grajeda, JP
PloS one. 2016;(10):e0163924
Abstract
BACKGROUND Although nowadays it is well known that the human transcriptome can importantly vary according to external or environmental condition, the reflection of this concept when studying oxidative stress and its direct relationship with gene expression profiling during the process of atherogenesis has not been thoroughly achieved. OBJECTIVE The ability to analyze genome-wide gene expression through transcriptomics has shown that the genome responds dynamically to diverse stimuli. Here, we describe the transcriptome of human vascular smooth muscle cells (hVSMC) stimulated by native and oxidized low-density lipoprotein (nLDL and oxLDL respectively), with the aim of assessing the early molecular changes that induce a response in this cell type resulting in a transcriptomic transformation. This expression has been demonstrated in atherosclerotic plaques in vivo and in vitro, particularly in the light of the oxidative modification hypothesis of atherosclerosis. APPROACH AND RESULTS Total RNA was isolated with TRIzol reagent (Life Technologies) and quality estimated using an Agilent 2100 bioanalyzer. The transcriptome of hVSMC under different experimental conditions (1,5 and 24 hours for nLDL and oxLDL) was obtained using the GeneChip Human Gene 1.0 ST (Affymetrix) designed to measure gene expression of 28,869 well-annotated genes. A fixed fold-change cut-off corresponding to ± 2 was used to identify genes exhibiting the most significant variation and statistical significance (P< 0.05), and 8 genes validated by qPCR using Taqman probes. CONCLUSIONS 10 molecular processes were significantly affected in hVSMC: Apoptosis and cell cycle, extracellular matrix remodeling, DNA repair, cholesterol efflux, cGMP biosynthesis, endocytic mechanisms, calcium homeostasis, redox balance, membrane trafficking and finally, the immune response to inflammation. The evidence we present supporting the hypothesis for the involvement of oxidative modification of several processes and metabolic pathways in atherosclerosis is strengthen by the fact that gene expression patterns obtained when hVSMC are incubated for a long period of time in the presence of nLDL, correspond very much the same as when cells are incubated for a short period of time in the presence of chemically modified oxLDL. Our results indicate that under physiological conditions and directly related to specific environmental conditions, LDL particles most probably suffer chemical modifications that initially serve as an alert signal to overcome a harmful stimulus that with time might get transformed to a pathological pattern and therefore consolidate a pathological condition.
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Direct effects of fatty meals and adiposity on oxidised low-density lipoprotein.
Laguna-Camacho, A, Alonso-Barreto, AS, Mendieta-Zerón, H
Obesity research & clinical practice. 2015;(3):298-300
Abstract
High-fat intake and high adiposity contribute to hyperlipaemia. In a hyperlipaemic state, lipoproteins infiltrate arterial wall where they are modified and cause an immune response characteristic of atherosclerosis. A small fraction of modified lipoproteins including oxidised low-density lipoprotein (ox-LDL) returns to circulation. The present study tracked high-fat meals during four weeks as to find effects of sustained frequency change on adiposity and ox-LDL. The findings indicated that changes in frequency of consumption of high-fat eating episodes correlated directly with changes in adiposity and ox-LDL. Hence the number of fatty meals consumed by people with overweight or obesity in few weeks could affect the atherogenic process.
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HELP LDL apheresis reduces plasma pentraxin 3 in familial hypercholesterolemia.
Zanetti, M, Zenti, M, Barazzoni, R, Zardi, F, Semolic, A, Messa, MG, Mearelli, F, Russi, G, Fonda, M, Scarano, L, et al
PloS one. 2014;(7):e101290
Abstract
BACKGROUND Pentraxin 3 (PTX3), a key component of the humoral arm of innate immunity, is secreted by vascular cells in response to injury, possibly aiming at tuning arterial activation associated with vascular damage. Severe hypercholesterolemia as in familial hypercholesterolemia (FH) promotes vascular inflammation and atherosclerosis; low-density lipoprotein (LDL) apheresis is currently the treatment of choice to reduce plasma lipids in FH. HELP LDL apheresis affects pro- and antiinflammatory biomarkers, however its effects on PTX3 levels are unknown. We assessed the impact of FH and of LDL removal by HELP apheresis on PTX3. METHODS Plasma lipids, PTX3, and CRP were measured in 19 patients with FH undergoing chronic HELP LDL apheresis before and after treatment and in 20 control subjects. In the patients assessment of inflammation and oxidative stress markers included also plasma TNFα, fibrinogen and TBARS. RESULTS At baseline, FH patients had higher (p = 0.0002) plasma PTX3 than matched control subjects. In FH PTX3 correlated positively (p≤0.05) with age, gender and CRP and negatively (p = 0.01) with HELP LDL apheresis vintage. The latter association was confirmed after correction for age, gender and CRP. HELP LDL apheresis acutely reduced (p≤0.04) plasma PTX3, CRP, fibrinogen, TBARS and lipids, but not TNFα. No association was observed between mean decrease in PTX3 and in LDL cholesterol. PTX3 paralleled lipids, oxidative stress and inflammation markers in time-course study. CONCLUSION FH is associated with increased plasma PTX3, which is acutely reduced by HELP LDL apheresis independently of LDL cholesterol, as reflected by the lack of association between change in PTX3 and in LDL levels. These results, together with the finding of a negative relationship between PTX3 and duration of treatment suggest that HELP LDL apheresis may influence both acutely and chronically cardiovascular outcomes in FH by modulating PTX3.
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Dietary grape powder increases IL-1β and IL-6 production by lipopolysaccharide-activated monocytes and reduces plasma concentrations of large LDL and large LDL-cholesterol particles in obese humans.
Zunino, SJ, Peerson, JM, Freytag, TL, Breksa, AP, Bonnel, EL, Woodhouse, LR, Storms, DH
The British journal of nutrition. 2014;(3):369-80
Abstract
Obese individuals are at an increased risk of developing CVD, hypertension, type 2 diabetes, and bacterial and viral infections when compared with the normal-weight population. In a 9-week randomised, double-blind, cross-over study, twenty-four obese subjects aged between 20 and 60 years and with a BMI between 30 and 45 kg/m2 were fed grape or placebo powder for 3-week intervals to determine the effects of dietary grapes on blood lipid profiles, plasma inflammatory marker concentrations and immune cell function. Blood samples were collected on days 1 and 8 for obtaining baseline information and at weeks 3, 4, 8 and 9. Comprehensive chemistry panels, lipid profile analyses by NMR, measurement of plasma inflammatory marker concentrations, and analyses of cytokine production by activated T lymphocytes and monocytes were performed for each blood draw. Dietary grape powder reduced the plasma concentrations of large LDL-cholesterol and large LDL particles compared with the placebo powder (P< 0·05). The concentrations of interferon-γ, TNF-α, IL-4 and IL-10 were measured in supernatants from peripheral blood mononuclear cells (PBMC) activated with anti-CD3/CD28 antibodies and those of TNF-α, IL-1β, IL-6 and IL-8 were measured in supernatants from PBMC activated with lipopolysaccharide (LPS). No difference in the production of T-cell cytokines was observed between the two intervention groups. The production of IL-1β and IL-6 was increased in supernatants from LPS-activated PBMC in the grape powder group compared with the placebo powder group (P< 0·05). These data suggest that dietary grapes may decrease atherogenic lipid fractions in obese individuals and increase the sensitivity of monocytes in a population at a greater risk of developing infections.
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Curcuminoids modulate pro-oxidant-antioxidant balance but not the immune response to heat shock protein 27 and oxidized LDL in obese individuals.
Sahebkar, A, Mohammadi, A, Atabati, A, Rahiman, S, Tavallaie, S, Iranshahi, M, Akhlaghi, S, Ferns, GA, Ghayour-Mobarhan, M
Phytotherapy research : PTR. 2013;(12):1883-8
Abstract
Curcuminoids have potentially important functional qualities including anti-inflammatory and antioxidant properties. In this randomized double-blind placebo-controlled cross-over trial, the effects of a curcuminoid supplement on serum pro-oxidant-antioxidant balance (PAB) and antibody titres to Hsp27 (anti-Hsp27) and oxLDL (anti-oxLDL) were investigated. Thirty obese individuals were randomized to receive either curcuminoids (1 g/day) or placebo for a period of 30 days. After a wash-out period of 2 weeks, subjects were crossed over to the alternate regimen for another 30 days. Serum PAB along with anti-Hsp27 and anti-oxLDL titres was measured at the beginning and at the end of each study period. There was no significant carry-over effect for any of the assessed parameters. Curcuminoid supplementation was associated with a significant decrease in PAB (p = 0.044). However, no significant change was observed in serum concentrations of anti-Hsp27 or anti-oxLDL (p > 0.05). These findings suggest that oral curcuminoids supplementation (1g/day) is effective in reducing oxidative stress burden, though this needs to be validated in larger study populations.
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Pathogenic role of modified LDL antibodies and immune complexes in atherosclerosis.
Lopes-Virella, MF, Virella, G
Journal of atherosclerosis and thrombosis. 2013;(10):743-54
Abstract
There is strong evidence supporting a key role of the adaptive immune response in atherosclerosis, given that both activated Th cells producing predominantly interferon-γ and oxidized LDL (oxLDL) and the corresponding antibodies have been isolated from atheromatous plaques. Studies carried out using immune complexes (IC) prepared with human LDL and rabbit antibodies have demonstrated proatherogenic and pro-inflammatory properties, mostly dependent on the engagement of Fcγ receptors Ⅰ and Ⅱ in macrophages and macrophage-like cell lines. Following the development of a methodology for isolating modified LDL (mLDL) antibodies from serum and isolated IC, it was confirmed that antibodies reacting with oxLDL and advanced glycation end product-modified LDL are predominantly IgG of subtypes 1 and 3 and that mLDL IC prepared with human reagents possesses pro-inflammatory and proatherogenic properties. In previous studies, LDL separated from isolated IC has been analyzed for its modifications, and the reactivity of antibodies isolated from the same IC with different LDL modifications has been tested. Recently, we obtained strong evidence suggesting that the effects of mLDL IC on phagocytic cells are modulated by the composition of the mLDL. Clinical studies have shown that the level of mLDL in circulating IC is a strong predictor of cardiovascular disease (CVD) and, in diabetic patients, other significant complications, such as nephropathy and retinopathy. In conclusion, there is convincing ex vivo and clinical data supporting the hypothesis that, in humans, the humoral immune response to mLDL is pathogenic rather than protective.