Co-Stimulation of PAFR and CD36 Is Required for oxLDL-Induced Human Macrophages Activation

The oxidative process of LDL particles generates molecules which are structurally similar to platelet-activating factor (PAF), and some effects of oxidized LDL (oxLDL) have been shown to be dependent on PAF receptor (PAFR) activation. In a previous study, we showed that PAFR is required for upregulation of CD36 and oxLDL uptake. In the present study we analyzed the molecular mechanisms activated by oxLDL in human macrophages and the contribution of PAFR to this response. Human adherent monocytes/macrophages were stimulated with oxLDL. Uptake of oxLDL and CD36 expression were determined by flow cytometry; MAP kinases and Akt phosphorylation by Western blot; IL-8 and MCP-1 concentration by ELISA and mRNA expression by real-time PCR. To investigate the participation of the PI3K/Akt pathway, G alpha i-coupled protein or PAFR, macrophages were treated with LY294002, pertussis toxin or with the PAFR antagonists WEB2170 and CV3988, respectively before addition of oxLDL. It was found that the addition of oxLDL to human monocytes/macrophages activates the PI3K/Akt pathway which in turn activates the MAPK (p38 and JNK). Phosphorylation of Akt requires the engagement of PAFR and a G alpha i-coupled protein. The upregulation of CD36 protein and the uptake of oxLDL as well as the IL-8 production are dependent on PI3K/Akt pathway activation. The increased CD36 protein expression is dependent on PAFR and G alpha i-coupled protein. Transfection studies using HEK 293t cells showed that oxLDL uptake occurs with either PAFR or CD36, but IL-8 production requires the co-transfection of both PAFR and CD36. These findings show that PAFR has a pivotal role in macrophages response to oxLDL and suggest that pharmacological intervention at the level of PAFR activation might be beneficial in atherosclerosis.

Uptake of oxLDL and IL-10 production by macrophages requires PAFR and CD36 recruitment into the same lipid rafts

Macrophage interaction with oxidized low-density lipoprotein (oxLDL) leads to its differentiation into foam cells and cytokine production, contributing to atherosclerosis development. In a previous study, we showed that CD36 and the receptor for platelet-activating factor (PAFR) are required for oxLDL to activate gene transcription for cytokines and CD36. Here, we investigated the localization and physical interaction of CD36 and PAFR in macrophages stimulated with oxLDL. We found that blocking CD36 or PAFR decreases oxLDL uptake and IL-10 production. OxLDL induces IL-10 mRNA expression only in HEK293T expressing both receptors (PAFR and CD36). OxLDL does not induce IL-12 production. The lipid rafts disruption by treatment with βCD reduces the oxLDL uptake and IL-10 production. OxLDL induces co-immunoprecipitation of PAFR and CD36 with the constitutive raft protein flotillin-1, and colocalization with the lipid raft-marker GM1-ganglioside. Finally, we found colocalization of PAFR and CD36 in macrophages from human atherosclerotic plaques. Our results show that oxLDL induces the recruitment of PAFR and CD36 into the same lipid rafts, which is important for oxLDL uptake and IL-10 production. This study provided new insights into how oxLDL interact with macrophages and contributing to atherosclerosis development.

The I405V and Taq1B polymorphisms of the CETP gene differentially affect sub-clinical carotid atherosclerosis

Background: Cholesteryl ester transfer protein (CETP) plays a major role in lipid metabolism, but studies on the association of CETP polymorphisms with risks of cardiovascular disease are inconsistent. This study investigated whether the CETP gene I405V and Taq1B polymorphisms modified subclinical atherosclerosis in an asymptomatic Brazilian population sample. Methods: The polymorphisms were analyzed using polymerase chain reaction in 207 adult volunteers. Serum lipid profiles, oxLDL Ab titers, C-reactive protein and tumor necrosis factor-a concentrations and CETP and phospholipid transfer protein (PLTP) activities were determined, and common carotid artery intima-media thickness (cIMT) was measured using ultrasonography. Results: No differences in cIMT were observed between the presence or absence of the minor B2 and V alleles in either polymorphism. However, inverse correlations between mean cIMT and CETP activity in the presence of these polymorphisms were observed, and positive correlations of these polymorphisms with PLTP activity and oxLDL Ab titers were identified. Moreover, logistic multivariate analysis revealed that the presence of the B2 allele was associated with a 5.1-fold (CI 95%, OR: 1.26 - 21.06) increased risk for cIMT, which was equal and above the 66th percentile and positively interacted with age. However, no associations with the V allele or CETP and PLTP activities were observed. Conclusions: None of the studied parameters, including CETP activity, explained the different relationships between these polymorphisms and cIMT, suggesting that other non-determined factors were affected by the genotypes and related to carotid atherosclerotic disease.

Association between diet and polymorphisms in individuals with statin-controlled dyslipidaemia grouped according to oxidative stress biomarkers

The objective of this study was to investigate whether differences in diet and in single-nucleotide polymorphisms (SNPs) found in paraoxonase-1 (PON-1), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), cholesterol ester transfer protein (CETP) and apolipoprotein E (APOE) genes, are associated with oxidative stress biomarkers and consequently with susceptibility of low-density cholesterol (LDL) to oxidation. A multivariate approach was applied to a group of 55 patients according to three biomarkers: plasma antioxidant activity, malondialdehyde and oxidized LDL (oxLDL) concentrations. Individuals classified in Cluster III showed the worst prognoses in terms of antioxidant activity and oxidative status. Individuals classified in Cluster I presented the lowest oxidative status, while individuals grouped in Cluster II presented the highest levels of antioxidant activity. No difference in nutrient intake was observed among the clusters. Significantly higher gamma- and delta-tocopherol concentrations were observed in those individuals with the highest levels of antioxidant activity. No single linear regression was statistically significant, suggesting that mutant alleles of the SNPs selected did not contribute to the differences observed in oxidative stress response. Although not statistically significant, the p value of the APO E coefficient for oxLDL response was 0.096, indicating that patients who carry the TT allele of the APO E gene tend to present lower plasma oxLDL concentrations. Therefore, the differences in oxidative stress levels observed in this study could not be attributed to diet or to the variant alleles of PON-1, CETP, HMGCR or APO E. This data supports the influence of gamma-tocopherol and delta-tocopherol on antioxidant activity, and highlights the need for further studies investigating APO E alleles and LDL oxidation.

Oxidized low-density lipoproteins and their antibodies: Relationships with the reverse cholesterol transport and carotid atherosclerosis in adults without cardiovascular diseases

Background: Metabolic predictors and the atherogenicity of oxidized LDL (oxLDL) and the specific antibodies against oxLDL (oxLDL Ab) are unclear and controversial. Methods: In 107 adults without atherosclerotic manifestations, we measured oxLDL and oxLDL Ab, and also the activities of CETP. PLTP, lipases and the carotid intima-media thickness (cIMT). Comparisons were performed for the studied parameters between the lowest and the highest tertile of oxLDL and oxLDL Ab, and the relationships between studied variables were evaluated. Results: Subjects with higher oxLDL Ab present reduced hepatic lipase activity and borderline increased cIMT. In the highest oxLDL tertile, besides the higher levels of total cholesterol, LDL-C and apoB100, we found reduced CETP activity and higher cIMT. A significant correlation between oxLDL Ab and cIMT, independent of oxLDL, and a borderline correlation between oxLDL and cIMT independent of oxLDL Ab were found. In the multivariate analysis, apoAl was a significant predictor of oxLDL Ab, in contrast to regulation of oxLDL by apoB100, PLTP and inverse of CETP. Conclusions: In adults without atherosclerotic disease, the metabolic regulation and carotid atherosclerosis of oxLDLAb and oxLDL groups, characterized a dual trait in oxLDL Ab, as a contributor to carotid atherosclerosis, much less so than oxidized LDL, and with a modest atheroprotective role. (C) 2012 Elsevier B.V. All rights reserved.

Association of postalimentary lipemia with atherosclerotic manifestations

We identified different lipemic and metabolic responses after the ingestion of a standardized meal by healthy adults and related them to atherosclerotic markers. Samples from 60 normolipidemic adults were collected before and after a liquid meal (40 g fat/m² body surface) at 0, 2, 4, 6, and 8 h for measurements of lipids, free fatty acids (FFA), insulin, cholesteryl ester transfer protein (CETP), autoantibodies to epitopes of oxidized LDL (oxLDL Ab), lipolytic activities, and apolipoprotein E polymorphism. Mean carotid intima-media thickness (cIMT) was determined by Doppler ultrasound. The volunteers were classified into early (N = 39) and late (N = 31) triacylglycerol (TAG) responders to the test meal. Late responders showed lower HDL cholesterol concentration at fasting and in the TAG peak, lower insulin and higher FFA concentrations compared to early responders. Multivariate regression analyses showed that mean cIMT was associated with gender (male) and age in early responders and by cholesterol levels at the 6th hour in late responders. oxLDL Ab were explained by lipoprotein lipase and negatively by hepatic lipase and oxLDL Ab (fasting period) by CETP (negative) and FFA (positive). This study is the first to identify a postalimentary insulin resistance state, combined with a reduced CETP response exclusively among late responders, and the identification of the regulators of postalimentary atherogenicity. Further research is required to determine the metabolic mechanisms described in the different postalimentary phenotypes observed in this study, as well as in different pathological states, as currently investigated in our laboratory.

Adaptive immunity is related to coronary artery disease severity after acute coronary syndrome in subjects with metabolic syndrome

Metabolic syndrome (MetS) is an inflammatory state associated with high coronary disease risk. Inflammation and adaptive immunity modulate atherosclerosis and plaque instability. We examined early changes in anti-oxidized lowdensity lipoprotein (LDL) (anti-oxLDL) autoantibodies (Abs) in patients with MetS after an acute coronary syndrome (ACS). Patients of both genders (n=116) with MetS were prospectively included after an acute yocardial infarction (MI) or hospitalization due to unstable angina. Anti-oxLDL Abs (IgG class) were assayed at baseline, three and six weeks after ACS. The severity of coronary disease was evaluated by the Gensini score. We observed a decrease in anti-oxLDL Abs titers (p<0.002 vs. baseline), mainly in males (p=0.01), in those under 65 y (p=0.03), and in subjects with Gensini score above median (p=0.04). In conclusion, early decrease in circulating anti-oxLDL Abs is associated with coronary disease severity among subjects with MetS.

Phage display identification of CD100 in human atherosclerotic plaque macrophages and foam cells

Atherosclerosis is a complex disease in which vessels develop plaques comprising dysfunctional endothelium, monocyte derived lipid laden foam cells and activated lymphocytes. Considering that humans and animal models of the disease develop quite distinct plaques, we used human plaques to search for proteins that could be used as markers of human atheromas. Phage display peptide libraries were probed to fresh human carotid plaques, and a bound phage homologous to plexin B1, a high affinity receptor for CD100, was identified. CD100 is a member of the semaphorin family expressed by most hematopoietic cells and particularly by activated T cells. CD100 expression was analyzed in human plaques and normal samples. CD100 mRNA and protein were analyzed in cultured monocytes, macrophages and foam cells. The effects of CD100 in oxLDL-induced foam cell formation and in CD36 mRNA abundance were evaluated. Human atherosclerotic plaques showed strong labeling of CD100/SEMA4D. CD100 expression was further demonstrated in peripheral blood monocytes and in in vitro differentiated macrophages and foam cells, with diminished CD100 transcript along the differentiation of these cells. Incubation of macrophages with CD100 led to a reduction in oxLDL-induced foam cell formation probably through a decrease of CD36 expression, suggesting for the first time an atheroprotective role for CD100 in the human disease. Given its differential expression in the numerous foam cells and macrophages of the plaques and its capacity to decrease oxLDL engulfment by macrophages we propose that CD100 may have a role in atherosclerotic plaque development, and may possibly be employed in targeted treatments of these atheromas.