Psychological Impact of the COVID-19 Pandemic on Dentists in Latin America's Epicenter: São Paulo, Brazil.

The present study aimed to assess the prevalence and associated factors of stress and anxiety symptoms among dentists during the COVID-19 pandemic in the state of São Paulo, Brazil. A structured questionnaire was sent electronically to 93,280 dentists with active registration in the Dental Council of São Paulo, Brazil, enquiring about information regarding the first-wave peak period in Brazil. Descriptive analyses of background characteristics, perceptions of preparedness, and psychological impact were calculated. Multiple logistic regression analysis was performed, and independent variables that showed p < 0.20 were used in the adjusted logistic regression model to compare the psychological impact on dental professionals. Among the 2113 respondents, female participants had 63% lower chance of reporting anxiety than males. Older dentists had a lower likelihood of reporting anxiety compared to 21-30-year-old dentists (p ≤ 0.05). Dentists working in the public health service were 1.78 times more likely to report anxiety than those who worked in private practice. Finally, dentists in the COVID-19 high-risk group and those with a family or team member with a positive COVID-19 diagnosis were more likely to have anxiety. This study can help dental and other healthcare professionals to better understand the consequences of COVID-19 in terms of mental health.

Kynurenine inhibits melanogenesis in human melanocyte-keratinocyte co-cultures and in a reconstructed 3D skin model.

Kynurenine (KYN), the most abundant metabolite of tryptophan, is classically associated with immune tolerance and tumor immune escape. In the last years, KYN is in the spotlight in other biological processes. Here, we showed that KYN inhibited tyrosinase expression and melanin content in primary human melanocyte and keratinocyte co-cultures. Furthermore, KYN decreased melanosome content in a 3D human skin reconstruction model. In these experiments, we used tyrosine + NH4 Cl to induce pigmentation. We compared the inhibitory effect of KYN on melanogenesis with the already known inhibitory effect promoted by IFN-γ. Since increased KYN production depends on the IFN-γ-inducible enzyme indoleamine-2,3-dioxygenase (IDO), we propose that part of the effect of IFN-γ on melanogenesis involves KYN production. From that, we tested if, during melanogenesis, changes in tryptophan metabolism would occur. For this purpose, we measured tryptophan, KYN and downstream products along with pigmentation. There were no significant changes in Trp metabolism, except for the high consumption of kynurenic acid. Our data identify the skin as a potential target for the action of KYN relevant for skin physiology and pigmentation. The results are discussed concerning the high production of KYN in skin inflammatory disorders and cancer.

Cost-effectiveness of ribociclib plus letrozole versus palbociclib plus letrozole or letrozole as monotherapy in first-line treatment of postmenopausal women with HR+/HER2- locally advanced or metastatic breast cancer: a Brazilian private payer perspective.

BACKGROUND: The global burden of breast cancer (BC) is high, especially in advanced stages. CDK 4/6 inhibitors represent a paradigm shift in the treatment of advanced BC HR+/HER2-, given the clinically and statistically significant gain in overall survival associated with this new class of medications. Nevertheless, as an innovation, the incorporation of these drugs impacts healthcare budgets, requiring cost-effectiveness analyses for decision-making. The aim of this study was to evaluate the cost-effectiveness of ribociclib plus letrozole compared with palbociclib plus letrozole or letrozole as monotherapy for first-line treatment of postmenopausal women with HR+/HER2- locally advanced or metastatic BC (aBC) from a Brazilian private healthcare system perspective. METHODS: A model including progression-free survival (PFS), progressed disease, and death health states was used to simulate lifetime costs and outcomes. PFS and overall survival were derived from the MONALEESA-2 trial (lifetime horizon). Healthcare costs included drug acquisition and monitoring, subsequent therapies, adverse events, and end-of-life costs. Effectiveness was measured in quality-adjusted life-years (QALYs). Deterministic and probabilistic sensitivity analyses were performed. RESULTS: The total cost of treatment with ribociclib plus letrozole was USD 72,091.82 versus USD 92,749.64 for palbociclib plus letrozole. Total QALYs were 3.30 and 3.16, respectively. Base-case analysis showed ribociclib as dominant over palbociclib in first-line treatment of women with HR+/HER2- aBC, associated with cost savings and QALY gains. The total cost of treatment with ribociclib plus letrozole was USD 83,058.73 versus USD 29,215.10 for letrozole. Total QALYs were 3.84 and 2.61, respectively. Compared with letrozole, ribociclib plus letrozole was associated with an incremental cost of USD 53,843.64 and an incremental QALY gain of 1.23, with incremental cost-effectiveness ratio of USD 43,826.91 per QALY gained. CONCLUSIONS: As demonstrated by the cost-effectiveness dominance over palbociclib, ribociclib results in savings when used as first-line treatment in postmenopausal women with HR+/HER2- aBC, warranting incorporation in the private healthcare system.

Inteligência artificial para classificação e auxílio na terapia tópica de queimaduras em tempo real / Artificial intelligence for classification and aid in topical burn therapy in real time

Objetivo: descrever o desenvolvimento e validação de uma tecnologia (aplicativo) que se embasou na inteligência artificial para classificação e auxílio na terapia tópica de queimaduras em tempo real. Método: Trata-se de uma pesquisa metodológica desenvolvida à luz do Design de Interação Participativo Centrado no Usuário. Resultado: Mostrou que o aplicativo se apresentou válido na avaliação de enfermeiras e médicos com experiência na área; possibilitou o diagnóstico automático por imagem da classificação do tipo de queimadura e a identificação dos tratamentos da queimadura classificada. Conclusão: O objetivo do estudo foi alcançado ao apresentar o desenvolvimento do aplicativo, bem como uma positiva avaliação dele por profissionais, que também fizeram sugestões, já incorporadas. (AU)

Objective: to describe the development and validation of a prototype for mobile device to assist professionals to topical treatment in patients with burns. Methodology: This is a methodological research developed in light of the User-Centered Participatory Interaction Design. Results: showed that the application proved to be valid in the evaluation of nurses and physicians with experience in the area, enabled the automatic diagnosis by image of the classification of the type of burn and the identification of the treatments of the classified burn. Conclusion: that the aim of the study was reached when presenting the application development, as well as a positive evaluation of it by professionals, who also made suggestions, already incorporated. (AU)

Objetivo: describir el desarrollo y validación de una tecnología (aplicación) que se basó en la inteligencia artificial para clasificación y auxilio en la terapia tópica de quemaduras en tiempo real. Metodología: Se trata de una investigación metodológica desarrollada a la luz del Diseño de Interacción Participativa Centrado en el Usuario. Resultados: Muestra que la aplicación se presentó válida en la evaluación de enfermeras y médicos con experiencia en el área; permitió el diagnóstico automático por imagen de la clasificación del tipo de quemadura y la identificación de los tratamientos de la quemadura clasificada. Conclusión: El objetivo del estudio fue alcanzado al presentar el desarrollo de la aplicación, así como una positiva evaluación de él por profesionales, que también hicieron sugerencias, ya incorporadas. (AU)

Dietary interesterified fat enriched with palmitic acid induces atherosclerosis by impairing macrophage cholesterol efflux and eliciting inflammation.

Interesterified fats are currently being used to replace trans fatty acids. However, their impact on biological pathways involved in the atherosclerosis development was not investigated. Weaning male LDLr-KO mice were fed for 16weeks on a high-fat diet (40% energy as fat) containing polyunsaturated (PUFA), TRANS, palmitic (PALM), palmitic interesterified (PALM INTER), stearic (STEAR) or stearic interesterified (STEAR INTER). Plasma lipids, lipoprotein profile, arterial lesion area, macrophage infiltration, collagen content and inflammatory response modulation were determined. Macrophage cholesterol efflux and the arterial expression of cholesterol uptake and efflux receptors were also performed. The interesterification process did not alter plasma lipid concentrations. Although PALM INTER did not increase plasma cholesterol concentration as much as TRANS, the cholesterol enrichment in the LDL particle was similar in both groups. Moreover, PALM INTER induced the highest IL-1ß, MCP-1 and IL-6 secretion from peritoneal macrophages as compared to others. This inflammatory response elicited by PALM INTER was confirmed in arterial wall, as compared to PALM. These deleterious effects of PALM INTER culminate in higher atherosclerotic lesion, macrophage infiltration and collagen content than PALM, STEAR, STEAR INTER and PUFA. These events can partially be attributed to a macrophage cholesterol accumulation, promoted by apoAI and HDL2-mediated cholesterol efflux impairment and increased Olr-1 and decreased Abca1 and Nr1h3 expressions in the arterial wall. Interesterified fats containing palmitic acid induce atherosclerosis development by promoting cholesterol accumulation in LDL particles and macrophagic cells, activating the inflammatory process in LDLr-KO mice.

New PPARγ partial agonist improves obesity-induced metabolic alterations and atherosclerosis in LDLr(-/-) mice.

Peroxisome proliferator-activated receptor gamma (PPARγ) regulates multiple pathways involved in the pathogenesis of obesity and atherosclerosis. Here, we evaluated the therapeutic potential of GQ-177, a new thiazolidinedione, on diet-induced obesity and atherosclerosis. The intermolecular interaction between PPARγ and GQ-177 was examined by virtual docking and PPAR activation was determined by reporter gene assay identifying GQ-177 as a partial and selective PPARγ agonist. For the evaluation of biological activity of GQ-177, low-density lipoprotein receptor-deficient (LDLr(-/-)) C57/BL6 mice were fed either a high fat diabetogenic diet (diet-induced obesity), or a high fat atherogenic diet, and treated with vehicle, GQ-177 (20mg/kg/day), pioglitazone (20mg/kg/day, diet-induced obesity model) or rosiglitazone (15mg/kg/day, atherosclerosis model) for 28 days. In diet-induced obesity mice, GQ-177 improved insulin sensitivity and lipid profile, increased plasma adiponectin and GLUT4 mRNA in adipose tissue, without affecting body weight, food consumption, fat accumulation and bone density. Moreover, GQ-177 enhanced hepatic mRNA levels of proteins involved in lipid metabolism. In the atherosclerosis mice, GQ-177 inhibited atherosclerotic lesion progression, increased plasma HDL and mRNA levels of PPARγ and ATP-binding cassette A1 in atherosclerotic lesions. GQ-177 acts as a partial PPARγ agonist that improves obesity-associated insulin resistance and dyslipidemia with atheroprotective effects in LDLr(-/-) mice.

Aerobic exercise training enhances the in vivo cholesterol trafficking from macrophages to the liver independently of changes in the expression of genes involved in lipid flux in macrophages and aorta.

BACKGROUND: Regular exercise prevents and regresses atherosclerosis by improving lipid metabolism and antioxidant defenses. Exercise ameliorates the reverse cholesterol transport (RCT), an antiatherogenic system that drives cholesterol from arterial macrophages to the liver for excretion into bile and feces. In this study we analyzed the role of aerobic exercise on the in vivo RCT and expression of genes and proteins involved in lipid flux and inflammation in peritoneal macrophages, aortic arch and liver from wild type mice. METHODS: Twelve-week-old male mice were divided into sedentary and trained groups. Exercise training was performed in a treadmill (15 m/min, 30 min/day, 5 days/week). Plasma lipids were determined by enzymatic methods and lipoprotein profile by fast protein liquid chromatography. After intraperitoneal injection of J774-macrophages the RCT was assessed by measuring the recovery of (3)H-cholesterol in plasma, feces and liver. The expression of liver receptors was determined by immunoblot, macrophages and aortic mRNAs by qRT-PCR. (14)C-cholesterol efflux mediated by apo A-I and HDL2 and the uptake of (3)H-cholesteryl oleoyl ether ((3)H-COE)-acetylated-LDL were determined in macrophages isolated from sedentary and trained animals 48 h after the last exercise session. RESULTS: Body weight, plasma lipids, lipoprotein profile, glucose and blood pressure were not modified by exercise training. A greater amount of (3)H-cholesterol was recovered in plasma (24 h and 48 h) and liver (48 h) from trained animals in comparison to sedentary. No difference was found in (3)H-cholesterol excreted in feces between trained and sedentary mice. The hepatic expression of scavenger receptor class B type I (SR-BI) and LDL receptor (B-E) was enhanced by exercise. We observed 2.8 and 1.7 fold rise, respectively, in LXR and Cyp7a mRNA in the liver of trained as compared to sedentary mice. Macrophage and aortic expression of genes involved in lipid efflux was not systematically changed by physical exercise. In agreement, (14)C-cholesterol efflux and uptake of (3)H-COE-acetylated-LDL by macrophages was similar between sedentary and trained animals. CONCLUSION: Aerobic exercise in vivo accelerates the traffic of cholesterol from macrophages to the liver contributing to prevention and regression of atherosclerosis, independently of changes in macrophage and aorta gene expression.

Glycated human serum albumin isolated from poorly controlled diabetic patients impairs cholesterol efflux from macrophages: an investigation by mass spectrometry.

Advanced glycation end-products impair ABCA-1-mediated cholesterol efflux by eliciting inflammation, the generation of reactive oxygen species and endoplasmatic reticulum (ER) stress. The glycation level of human serum albumin (HSA) from type 1 and type 2 diabetic patients was determined by matrix assisted laser desorption/ionization (MALDI) mass spectrometry and related to possible impairment of ER function and cellular cholesterol efflux. Comparison of the MALDI spectra from healthy and diabetic subjects allowed us to determine an increased HSA mean mass of 1297 Da for type 1 and 890 Da for type 2. These values reflect a mean condensation of at least 8 glucose units and 5 glucose units, respectively. Mouse peritoneal macrophages were treated with HSA from control, type 1 and type 2 diabetic subjects in order to measure the expression of Grp78, Grp94, protein disulfide isomerase (PDI), calreticulin (CRT) and ABCA-1. (14)C-cholesterol overloaded-J774 macrophages were treated with HSA from control and diabetic subjects and further incubated with apo A-1 to determine the cholesterol efflux. Combined analyses comprising HSA from type 1 and type 2 diabetic patients were performed in cellular functional assays. In macrophages, PDI expression increased 89% and CRT 3.4 times in comparison to HSA from the control subjects. ABCA-1 protein level and apo A-I mediated cholesterol efflux were, respectively, 50% and 60% reduced in macrophages exposed to HSA from type 1 and type 2 diabetic patients when compared to that exposed to HSA from control subjects. We provide evidence that the level of glycation that occurs in albumin in vivo damages the ER function related to the impairment in macrophage reverse cholesterol transport and so contributes to atherosclerosis in diabetes.

In type 2 diabetes mellitus glycated albumin alters macrophage gene expression impairing ABCA1-mediated cholesterol efflux.

Advanced glycation end products (AGE) are elevated in diabetes mellitus (DM) and predict the development of atherosclerosis. AGE-albumin induces oxidative stress, which is linked to a reduction in ABCA-1 and cholesterol efflux. We characterized the glycation level of human serum albumin (HSA) isolated from poorly controlled DM2 (n = 11) patients compared with that of control (C, n = 12) individuals and determined the mechanism by which DM2-HSA can interfere in macrophage lipid accumulation. The HSA glycation level was analyzed by MALDI/MS. Macrophages were treated for 18 h with C- or DM2-HSA to measure the (14) C-cholesterol efflux, the intracellular lipid accumulation and the cellular ABCA-1 protein content. Agilent arrays (44000 probes) were used to analyze gene expression, and the differentially expressed genes were validated by real-time RT-PCR. An increased mean mass was observed in DM2-HSA compared with C-HSA, reflecting the condensation of at least 5 units of glucose. The cholesterol efflux mediated by apo AI, HDL3 , and HDL2 was impaired in DM2-HSA-treated cells, which was related to greater intracellular lipid accumulation. DM2-HSA decreased Abcg1 mRNA expression by 26%. Abca1 mRNA was unchanged, although the final ABCA-1 protein content decreased. Compared with C-HAS-treated cells, NADPH oxidase 4 mRNA expression increased in cells after DM2-HSA treatment. Stearoyl-Coenzyme A desaturase 1, janus kinase 2, and low density lipoprotein receptor mRNAs were reduced by DM2-HSA. The level of glycation that occurs in vivo in DM2-HSA-treated cells selectively alters macrophage gene expression, impairing cholesterol efflux and eliciting intracellular lipid accumulation, which contribute to atherogenesis, in individuals with DM2.

N-acetylcysteine prevents endoplasmic reticulum stress elicited in macrophages by serum albumin drawn from chronic kidney disease rats and selectively affects lipid transporters, ABCA-1 and ABCG-1.

In chronic kidney disease (CKD) nontraditional risk factors, such as oxidative stress and advanced glycation end products (AGE) contribute to cardiovascular disease. Particularly, disturbances in reverse cholesterol transport favor the development of atherosclerosis. We analyzed the influence of N-acetylcysteine (NAC) in CKD rats on plasma concentration of lipid peroxides (TBARS) and AGE and on the impact of serum albumin in the development of macrophage endoplasmic reticulum stress (ERS) and cholesterol efflux, namely apo A-I and HDL2-mediated cholesterol removal and ABCA-1 and ABCG-1 protein level. CKD was induced by 5/6 nephrectomy in 2-month old male Wistar rats. Controls (Sham) were false operated. Animals were treated or not with NAC (600 mg/L of water). After 60 days serum albumin was isolated by FPLC and purified by alcoholic extraction. J774 macrophages were incubated with serum albumin (1 mg/mL; 18 h) from all groups, and the expression of ERS markers (protein disulfide isomerase - PDI, Grp78 and Grp94), ABCA-1 and ABCG-1 determined by immunoblot. HDL2 or apo A-I were used for cholesterol efflux assays. Protein and lipid composition of total HDL from Sham and CKD was determined and these particles tested on their abilities to accept cell cholesterol. Comparisons were done by one-way ANOVA and Newman Keuls post test. After 60 days of CKD, body weight was 10% lower in CKD compared to Sham (p < 0.01). This was prevented by NAC. Urea, creatinine, total cholesterol (TC), triglycerides (TG) (mg/dL), proteinuria (mg/24 h) (Sham, n = 31; Sham + NAC, n = 20; CKD, n = 74; CKD + NAC, n = 32), total AGE and pentosidine (n = 8; fluorescence arbitrary unit) and TBARS (n = 7; nmoL/mL) were higher in CKD (122 ± 8; 0.9 ± 0.07; 151 ± 6; 83 ± 4; 46 ± 2.5; 32,620 ± 673; 16,700 ± 1,370; 6.6 ± 0.5, respectively) and in CKD + NAC (91.4 ± 5; 0.6 ± 0.02; 126 ± 7.5; 73 ± 6; 51 ± 3.5; 24,720 ± 1,114; 10,080 ± 748; 4.5 ± 0.5, respectively) in comparison to Sham (41 ± 0.9; 0.4 ± 0.03; 76 ± 2.7; 51.5 ± 3; 14 ± 0.9; 21,750 ± 960; 5,314 ± 129; 2.0 ± 0.2, respectively; p < 0.001) and Sham + NAC (40 ± 0.9; 0.3 ± 0.02; 76 ± 2.6; 68 ± 4; 18.4 ± 1.5; 20,040 ± 700; 5,050 ± 267; 1.8 ± 0.2, respectively; p < 0.001). TC, urea, creatinine, total AGE, pentosidine and TBARS were respectively, 17%, 25%, 33%, 24%, 40% and 28% (p < 0.01) lower in CKD + NAC, than in CKD. Glycemia was higher in Sham + NAC (107 ± 4.6) and CKD + NAC (107 ± 2.6) than in Sham (96 ± 1.8; p < 0.05) and CKD (98 ± 1.6; p < 0.01), respectively. In macrophages (n = 6), CKD albumin increased PDI (3 and 6 times, p < 0.01) and Grp94 (66% and 80%, p < 0.01) in comparison to Sham and CKD + NAC-albumin treated cells, respectively. ABCA-1 expression was lower (87% and 70%, p < 0.001) in macrophage treated with Sham + NAC and CKD albumin respectively in comparison to Sham albumin; ABCG-1 was higher (4 and 7 times, p < 0.001) in macrophages treated with Sham + NAC and CKD + NAC albumin, respectively in comparison to Sham and CKD albumin. Apo A-I mediated cholesterol efflux was lower (59% and 70%, p < 0.0001) in macrophage treated with Sham + NAC and CKD albumin respectively in comparison to Sham albumin, however, the HDL2 mediated cholesterol efflux was higher (54% and 25%, p < 0.0001) in macrophage treated with Sham + NAC albumin, in comparison to Sham and CKD + NAC albumin, respectively. CKD-HDL was enriched in total protein and lipids compared to Sham-HDL but preserved its capacity to remove cholesterol from macrophages. NAC reduces plasma lipid peroxidation and AGE and abrogates ERS induced by CKD-albumin. Despite diminishing ABCA-1, NAC increases ABCG-1 that counteracts the reduction in apo A-I-mediated cholesterol efflux. NAC may contribute to attenuate the deleterious effects of CKD-albumin on lipid accumulation in macrophages helping to prevent atherogenesis in CKD.

The impact of dietary fatty acids on macrophage cholesterol homeostasis.

The impact of dietary fatty acids in atherosclerosis development may be partially attributed to their effect on macrophage cholesterol homeostasis. This process is the result of interplay between cholesterol uptake and efflux, which are permeated by inflammation and oxidative stress. Although saturated fatty acids (SAFAs) do not influence cholesterol efflux, they trigger endoplasmic reticulum stress, which culminates in increased lectin-like oxidized LDL (oxLDL) receptor (LOX1) expression and, consequently, oxLDL uptake, leading to apoptosis. Unsaturated fatty acids prevent most SAFAs-mediated deleterious effects and are generally associated with reduced cholesterol efflux, although α-linolenic acid increases cholesterol export. Trans fatty acids increase macrophage cholesterol content by reducing ABCA-1 expression, leading to strong atherosclerotic plaque formation. As isomers of conjugated linoleic acid (CLAs) are strong PPAR gamma ligands, they induce cluster of differentiation (CD36) expression, increasing intracellular cholesterol content. Considering the multiple effects of fatty acids on intracellular signaling pathways, the purpose of this review is to address the role of dietary fat in several mechanisms that control macrophage lipid content, which can determine the fate of atherosclerotic lesions.

Advanced glycated albumin impairs HDL anti-inflammatory activity and primes macrophages for inflammatory response that reduces reverse cholesterol transport.

OBJECTIVE: We investigated the effect of advanced glycated albumin (AGE-albumin) on macrophage sensitivity to inflammation elicited by S100B calgranulin and lipopolysaccharide (LPS) and the mechanism by which HDL modulates this response. We also measured the influence of the culture medium, isolated from macrophages treated with AGE-albumin, on reverse cholesterol transport (RCT). METHODS AND RESULTS: Macrophages were incubated with control (C) or AGE-albumin in the presence or absence of HDL, followed by incubations with S100B or LPS. Also, culture medium obtained from cells treated with C- or AGE-albumin, following S100B or LPS stimulation was utilized to treat naive macrophages in order to evaluate cholesterol efflux and the expression of HDL receptors. In comparison with C-albumin, AGE-albumin, promoted a greater secretion of cytokines after stimulation with S100B or LPS. A greater amount of cytokines was also produced by macrophages treated with AGE-albumin even in the presence of HDL. Cytokine-enriched medium, drawn from incubations with AGE-albumin and S100B or LPS impaired the cholesterol efflux mediated by apoA-I (23% and 37%, respectively), HDL(2) (43% and 47%, respectively) and HDL(3) (20% and 8.5%, respectively) and reduced ABCA-1 protein level (16% and 26%, respectively). CONCLUSIONS: AGE-albumin primes macrophages for an inflammatory response impairing the RCT. Moreover, AGE-albumin abrogates the anti-inflammatory role of HDL, which may aggravate the development of atherosclerosis in DM.

ER stress is associated with reduced ABCA-1 protein levels in macrophages treated with advanced glycated albumin - reversal by a chemical chaperone.

ATP-binding cassette transporter A1 mediates the export of excess cholesterol from macrophages, contributing to the prevention of atherosclerosis. Advanced glycated albumin (AGE-alb) is prevalent in diabetes mellitus and is associated with the development of atherosclerosis. Independently of changes in ABCA-1 mRNA levels, AGE-alb induces oxidative stress and reduces ABCA-1 protein levels, which leads to macrophage lipid accumulation. These metabolic conditions are known to elicit endoplasmic reticulum (ER) stress. We sought to determine if AGE-alb induces ER stress and unfolded protein response (UPR) in macrophages and how disturbances to the ER could affect ABCA-1 content and cholesterol efflux in macrophages. AGE-alb induced a time-dependent increase in ER stress and UPR markers. ABCA-1 content and cellular cholesterol efflux were reduced by 33% and 47%, respectively, in macrophages treated with AGE-alb, and both were restored by treatment with 4-phenyl butyric acid (a chemical chaperone that alleviates ER stress), but not MG132 (a proteasome inhibitor). Tunicamycin, a classical ER stress inductor, also impaired ABCA-1 expression and cholesterol efflux (showing a decrease of 61% and 82%, respectively), confirming the deleterious effect of ER stress in macrophage cholesterol accumulation. Glycoxidation induces macrophage ER stress, which relates to the reduction in ABCA-1 and in reverse cholesterol transport, endorsing the adverse effect of macrophage ER stress in atherosclerosis. Thus, chemical chaperones that alleviate ER stress may represent a useful tool for the prevention and treatment of atherosclerosis in diabetes.

Inhibition of macrophage oxidative stress prevents the reduction of ABCA-1 transporter induced by advanced glycated albumin.

We investigated the role of aminoguanidine and benfotiamine on the inhibition of reactive oxygen species (ROS) generation in macrophages induced by advanced glycated albumin (AGE-albumin) and its relationship with cell cholesterol homeostasis, emphasizing the expression of the ATP binding cassette transporter A-1 (ABCA-1). AGE-albumin was made by incubating fatty acid-free albumin with 10 mM glycolaldehyde. ROS production and ABCA-1 protein level were determined by flow cytometry in J774 macrophages treated along time with control (C) or AGE-albumin alone or in the presence of aminoguanidine or benfotiamine. Mitochondrial function was evaluated by oxygraphy. Compared to C-albumin, AGE-albumin increased ROS production in macrophages, which was ascribed to the activities of NADPH oxidase and of the mitochondrial system. Mitochondrial respiratory chain activity was reduced in cells incubated with AGE-albumin. ROS generation along time was associated with the reduction in macrophage ABCA-1 protein level. Aminoguanidine prevented ROS elevation and restored the ABCA-1 content in macrophages; on the other hand, benfotiamine that promoted a lesser reduction in ROS generation was not able to restore ABCA-1 levels. Inhibition of oxidative stress induced by AGE-albumin prevents disturbances in reverse cholesterol transport by curbing the reduction of ABCA-1 elicited by advanced glycation in macrophages and therefore may contribute to the prevention of atherosclerosis in diabetes mellitus.

Inibição do estresse do retículo endoplasmático restaura o conteúdo de ABCA-1 e o efluxo de colesterol em macrófagos tratados com albumina modificada por glicação avançada / Inhibition of endoplasmic reticulum stress restores the ABCA-1 protein level and cholesterol efflux in advanced glycated albumin-treated macrophages

Produtos de glicação avançada (AGE) prejudicam o metabolismo de lipoproteínas e o transporte reverso de colesterol, o que contribui para a aterosclerose no diabete melito (DM). Em particular, a albumina modificada por AGE (albumina-AGE) reduz a remoção de colesterol por diminuir o conteúdo do receptor ABCA-1 em macrófagos. Isto se vincula ao insulto oxidativo e inflamatório, os quais são indutores do estresse do retículo endoplasmático (RE). O objetivo do presente estudo foi avaliar, em macrófagos, os efeitos do tratamento com albumina-AGE sobre o estresse do RE e suas vias adaptativas (UPR), relacionando-os com o prejuízo na expressão do ABCA-1 e efluxo de colesterol celular. Albumina-AGE foi produzida pela incubação de albumina isenta em ácidos graxos com glicolaldeído 10 mM e, albumina controle (albumina-C) com PBS apenas. Albumina foi isolada do soro de pacientes portadores de DM com controle glicêmico inadequado (albumina-DM) ou indivíduos controles (albumina não- DM) por cromatografia para separação rápida de proteínas seguida por purificação alcoólica. Macrófagos de peritônio de camundongos ou macrófagos da linhagem J774 foram tratados com os diferentes tipos de albumina na presença ou ausência de ácido fenil butírico (PBA; chaperona química que alivia o estresse do RE) ou MG-132 (inibidor do sistema proteasomal) por diferentes intervalos de tempo. A expressão de marcadores do estresse do RE, UPR, proteína dissulfeto isomerase (PDI), calreticulina e ubiquitina foi determinada por imunoblot e o conteúdo de ABCA-1, por citometria de fluxo e imunocitoquímica. O efluxo de 14Ccolesterol foi avaliado, utilizando-se apoA-I como aceptora de colesterol. A albumina-AGE induziu aumento tempo-dependente na expressão das chaperonas marcadoras do estresse do RE - Gr78 e Grp94 - e de proteínas da UPR (ATF6 e eIF2-P) em comparação à albumina-C. O conteúdo de ABCA-1 e o efluxo de colesterol foram reduzidos em, respectivamente, 33% e 47% e ambos foram restaurados pelo...

Advanced glycation end products (AGE) disturb lipoprotein metabolism and reverse cholesterol transport, contributing to atherosclerosis in diabetes mellitus (DM). Particularly, advanced glycated albumin (AGE-albumin) reduces cell cholesterol removal by impairing the expression of ABCA-1 in macrophages. This is ascribed to the oxidative and inflammatory stress, conditions that elicit endoplasmic reticulum (ER) stress. In this study it was investigated the effect of AGE-albumin on ER stress and adaptative pathways (UPR) development in macrophages, and its relationship to the reduction in ABCA-1 expression and cholesterol efflux. AGE-albumin was prepared by incubating fatty acid free albumin with 10 mM glycolaldehyde and control albumin (C-albumin) with PBS only. Albumin was isolated from poorly controlled DM patients (DM-albumin) and control individuals (nonDMalbumin) by fast liquid chromatography and purified by alchoolic extraction. Mouse peritoneal macrophages or J774 cells were treated along time with the different types of albumin in the absence or presence of phenyl butiric acic (PBA; a chaperone that aleviates ER stress) or MG132 (a proteasomal inhibitor). The expression of ER stress and UPR markers, protein disulfide isomerase (PDI), calreticulin and ubiquitin was determined by immunoblot and ABCA-1 protein level, by flow cytometry and imunocytochemistry. 14Ccholesterol efflux was evaluated utilizing apo A-I as cholesterol acceptor. AGE-albumin induced a time-dependent increase in the expression of ER stress chaperone markers - Gr78 and Grp94 - and UPR proteins (ATF6 and eIF2-P) in comparison to C-albumin. ABCA-1 content and cholesterol efflux were diminished by, respectively, 33% and 47% and both were recovered by the treatment with PBA. The association between ER stress and ABCA-1 reduction was confirmed by the reduction, induced by tunicanycin (a classical ER stress inductior) in ABCA-1 protein level (61%) and cholesterol efflux (82%). AGE-albumin...

Advanced glycation in macrophages induces intracellular accumulation of 7-ketocholesterol and total sterols by decreasing the expression of ABCA-1 and ABCG-1.

BACKGROUND: Advanced glycation end products (AGE) alter lipid metabolism and reduce the macrophage expression of ABCA-1 and ABCG-1 which impairs the reverse cholesterol transport, a system that drives cholesterol from arterial wall macrophages to the liver, allowing its excretion into the bile and feces. Oxysterols favors lipid homeostasis in macrophages and drive the reverse cholesterol transport, although the accumulation of 7-ketocholesterol, 7alpha- hydroxycholesterol and 7beta- hydroxycholesterol is related to atherogenesis and cell death. We evaluated the effect of glycolaldehyde treatment (GAD; oxoaldehyde that induces a fast formation of intracellular AGE) in macrophages overloaded with oxidized LDL and incubated with HDL alone or HDL plus LXR agonist (T0901317) in: 1) the intracellular content of oxysterols and total sterols and 2) the contents of ABCA-1 and ABCG-1. METHODS: Total cholesterol and oxysterol subspecies were determined by gas chromatography/mass spectrometry and HDL receptors content by immunoblot. RESULTS: In control macrophages (C), incubation with HDL or HDL + T0901317 reduced the intracellular content of total sterols (total cholesterol + oxysterols), cholesterol and 7-ketocholesterol, which was not observed in GAD macrophages. In all experimental conditions no changes were found in the intracellular content of other oxysterol subspecies comparing C and GAD macrophages. GAD macrophages presented a 45% reduction in ABCA-1 protein level as compared to C cells, even after the addition of HDL or HDL + T0901317. The content of ABCG-1 was 36.6% reduced in GAD macrophages in the presence of HDL as compared to C macrophages. CONCLUSION: In macrophages overloaded with oxidized LDL, glycolaldehyde treatment reduces the HDL-mediated cholesterol and 7-ketocholesterol efflux which is ascribed to the reduction in ABCA-1 and ABCG-1 protein level. This may contribute to atherosclerosis in diabetes mellitus.