PPARγ partial agonist LPSF/GQ-16 prevents dermal and pulmonary fibrosis in HOCl-induced systemic sclerosis (SSc) and modulates cytokine production in PBMC of SSc patients.

Thiazolidinediones (TZD) are synthetic molecules that have a range of biological effects, including antifibrotic and anti-inflammatory, and they may represent a promising therapeutic strategy for systemic sclerosis (SSc). The aim of this study was to investigate the immunomodulatory and antifibrotic properties of LPSF/GQ-16, a TZD derivative, in peripheral blood mononuclear cells (PBMC) from SSc patients and in a murine model of SSc HOCl-induced. The PBMC of 20 SSc patients were stimulated with phytohemagglutinin (PHA) and treated with LPSF/GQ-16 for 48 h, later cytokines in the culture supernatants were quantified by sandwich enzyme-linked immunosorbent assay (ELISA) or cytometric bead array (CBA). Experimental SSc was induced by intradermal injections of hypochlorous acid (HOCl) for 6 weeks. HOCl-induced SSc mice received daily treatment with LPSF/GQ-16 (30 mg/kg) through intraperitoneal injections during the same period. Immunological parameters were evaluated by flow cytometry and ELISA, and dermal and pulmonary fibrosis were evaluated by RT-qPCR, hydroxyproline dosage and histopathological analysis. In PBMC cultures, it was possible to observe that LPSF/GQ-16 modulated the secretion of cytokines IL-2 (p < 0.001), IL-4 (p < 0.001), IL-6 (p < 0.001), IL-17A (p = 0.006), TNF (p < 0.001) and IFN-γ (p < 0.001). In addition, treatment with LPSF/GQ-16 in HOCl-induced SSc mice promoted a significant reduction in dermal thickening (p < 0.001), in the accumulation of collagen in the skin (p < 0.001), down-regulated the expression of fibrosis markers in the skin (Col1a1, α-Sma and Tgfß1, p < 0.001 for all) and lungs (Il4 and Il13, p < 0.001 for both), as well as reduced activation of CD4 + T cells (p < 0.001), B cells (p < 0.001) and M2 macrophages (p < 0.001). In conclusion, LPSF/GQ-16 showed immunomodulatory and antifibrotic properties, demonstrating the therapeutic potential of this molecule for SSc.

Genetic Deficiency of Indoleamine 2,3-dioxygenase Aggravates Vascular but Not Liver Disease in a Nonalcoholic Steatohepatitis and Atherosclerosis Comorbidity Model.

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease that increases cardiovascular disease risk. Indoleamine 2,3-dioxygenase-1 (IDO1)-mediated tryptophan (Trp) metabolism has been proposed to play an immunomodulatory role in several diseases. The potential of IDO1 to be a link between NASH and cardiovascular disease has never been investigated. Using Apoe-/-and Apoe-/-Ido1-/- mice that were fed a high-fat, high-cholesterol diet (HFCD) to simultaneously induce NASH and atherosclerosis, we found that Ido1 deficiency significantly accelerated atherosclerosis after 7 weeks. Surprisingly, Apoe-/-Ido1-/- mice did not present a more aggressive NASH phenotype, including hepatic lipid deposition, release of liver enzymes, and histopathological parameters. As expected, a lower L-kynurenine/Trp (Kyn/Trp) ratio was found in the plasma and arteries of Apoe-/-Ido1-/- mice compared to controls. However, no difference in the hepatic Kyn/Trp ratio was found between the groups. Hepatic transcript analyses revealed that HFCD induced a temporal increase in tryptophan 2,3-dioxygenase (Tdo2) mRNA, indicating an alternative manner to maintain Trp degradation during NASH development in both Apoe-/- and Apoe-/-Ido1-/mice-. Using HepG2 hepatoma cell and THP1 macrophage cultures, we found that iron, TDO2, and Trp degradation may act as important mediators of cross-communication between hepatocytes and macrophages regulating liver inflammation. In conclusion, we show that Ido1 deficiency aggravates atherosclerosis, but not liver disease, in a newly established NASH and atherosclerosis comorbidity model. Our data indicate that the overexpression of TDO2 is an important mechanism that helps in balancing the kynurenine pathway and inflammation in the liver, but not in the artery wall, which likely determined disease outcome in these two target tissues.

Reduced plasma zinc levels, lipid peroxidation, and inflammation biomarkers levels in hemodialysis patients: implications to cardiovascular mortality.

Despite the fact that low plasma zinc (Zn) levels play important roles in the oxidative stress, the relationships between lipid peroxidation and inflammation biomarkers with low plasma Zn levels have not been investigated in chronic kidney disease (CKD) patients. The aim of this study was to evaluate the Zn plasma levels, electronegative LDL [LDL(-)] levels, and inflammation markers as predictors of cardiovascular (CV) mortality in hemodialysis (HD) patients. Forty-five HD patients (28 men, 54.2 ± 12.7 years, 62.2 ± 51.4 months on dialysis and BMI 24.3 ± 4.1 kg/m(2)) were studied and compared to 20 healthy individuals (9 men, 51.6 ± 15.6 years, BMI 25.2 ± 3.9 kg/m(2)) and followed for 24 months to investigate the risks for CV mortality. LDL(-) levels were measured by ELISA, plasma Zn levels by atomic absorption spectrophotometry, C-reactive protein (CRP) level by immunoturbidimetric method, and tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), and plasminogen activator inhibitor-1 (PAI-1) levels by a multiplex assay kit. HD patients presented low plasma Zn levels (54.9 ± 16.1 µg/dL) and high-LDL(-) (0.18 ± 0.12 U/L) and TNF-α (5.5 ± 2.2 pg/mL) levels when compared to healthy subjects (78.8 ± 9.4µ g/dL, 0.10 ± 0.08U/L, 2.4 ± 1.1 pg/mL, respectively, p < 0.05). Zn plasma levels were negatively correlated to TNF-α (r = -0.49; p = 0.0001) and LDL(-) (r = -0.33; p = 0.008). During the 2 years, 24.4% of the patients died, all due to CV disease. Analysis by the Cox model showed that high CRP, TNF-α, IL-6 levels, and long duration of HD were significant predictors of mortality. In conclusion, reduced Zn levels were associated with lipid peroxidation and inflammation, and we confirm here in a Brazilian cohort of HD patients that inflammation markers are strong predictors of CV death.

Association between circulating electronegative low-density lipoproteins and serum ferritin in hemodialysis patients: a pilot study.

BACKGROUND: Iron supplementation is a common recommendation to chronic kidney disease patients undergoing hemodialysis (HD). However, iron excess is closely associated with lipid peroxidation and, it is well known that electronegative low-density lipoproteins (LDL[-]) are present at higher plasma concentrations in diseases with high cardiovascular risk such as chronic kidney disease. Thus, the aim of this study was to investigate whether ferritin levels are associated with LDL(-) levels in HD patients. DESIGN: This was a cross-sectional study. SETTING: This study was conducted from a private clinic in Rio de Janeiro, Brazil. PATIENTS: The study included 27 HD patients and 15 healthy subjects. METHODS AND PROCEDURES: Twenty-seven HD patients (14 men, 58.6 ± 10 years, 62.2 ± 51.4 months on dialysis, and body mass index: 24.4 ± 4.2 kg/m(2)) were studied and compared with 15 healthy individuals (6 men, 53.8 ± 15.4 years, body mass index: 24.5 ± 4.3 kg/m(2)). Serum LDL(-) levels were measured using the enzyme-linked immunosorbent assay method; ferritin levels by commercially available kits, and tumor necrosis factor-α, interleukin-6, monocyte chemoattractant protein-1, and plasminogen activator inhibitor-1 were determined with a multiplex assay kit manufactured by R&D Systems. RESULTS: The HD patients presented higher LDL(-) and tumor necrosis factor-α levels (0.15 ± 0.13 U/L and 5.9 ± 2.3 pg/mL, respectively) than healthy subjects (0.07 ± 0.05 U/L and 2.3 ± 1.3 pg/mL, respectively) (P = .0001). The mean ferritin level in HD patients was 1,117.5 ± 610.4 ng/mL, and 90% of patients showed ferritin levels exceeding 500 ng/mL. We found a positive correlation between LDL(-) and ferritin in the patients (r = 0.48; P = .01), and ferritin was a significant contributor to LDL(-) concentrations independent of inflammation. CONCLUSIONS: Excess body iron stores for HD patients was associated with signs of increased oxidative stress, as reflected by increased LDL(-) levels in HD patients.

Ischemia-reperfusion damage is attenuated by GQ-11, a peroxisome proliferator-activated receptor (PPAR)-α/γ agonist, after aorta clamping in rats.

INTRODUCTION: Ischemia-Reperfusion (I/R) damage is one of the major challenges in cardiothoracic surgeries and in a pathological manner, is identified by exacerbated damage signals resulted from blood supply restriction and subsequent flow restoration and re­oxygenation. I/R damage includes cellular dysfunction and death, impairing tissue and organ function. Inflammation and oxidative stress are known to underlie either ischemia or reperfusion, leaded by HIF, TNF-α, NF-κB, IL-6 and ROS formation. However, the available approaches to prevent I/R damage has been unsuccessful so far. As agonists of peroxisome-proliferation activation receptor (PPAR) are described as transcription factors related to anti-inflammatory factors, we proposed to observe the effects of novel dual agonist, GQ-11, in I/R-related damage. METHODS: Male, Wistar rats, 60 days age and 305 g body weight average were treated with vehicle, pioglitazone or GQ-11 (20 mg/kg) for 7 consecutive days and were submitted to aorta clamping for 30 min followed by 3 h of reperfusion. 18F-fluorodeoxyglucose (18F-FDG), an analog of glucose associated with inflammation when accumulated, was observed in liver and bowel by positron emission tomography (PET). RESULTS: GQ-11 decreased 18F-FDG uptake in liver and bowel when compared to vehicle and pioglitazone. The treatment also modulated inflammatory markers IL-10, TGF-ß, IL-6, IL1-ß, TNFα, and CCL-2, besides antioxidant enzymes such as catalase, GPx and SOD. CONCLUSION: Inflammation and oxidative stress showed to be important processes to be regulated in I/R in order to prevent exacerbated responses that leads to cell/tissue dysfunction and death. PPAR agonists - including GQ-11 - might be promising agents in a strategy to avoid tissue dysfunction and death after cardiothoracic surgeries.

scFv-Anti-LDL(-)-Metal-Complex Multi-Wall Functionalized-Nanocapsules as a Promising Tool for the Prevention of Atherosclerosis Progression.

Atherosclerosis can be originated from the accumulation of modified cholesterol-rich lipoproteins in the arterial wall. The electronegative LDL, LDL(-), plays an important role in the pathogenesis of atherosclerosis once this cholesterol-rich lipoprotein can be internalized by macrophages, contributing to the formation of foam cells, and provoking an immune-inflammatory response. Herein, we engineered a nanoformulation containing highly pure surface-functionalized nanocapsules using a single-chain fragment variable (scFv) reactive to LDL(-) as a ligand and assessed whether it can affect the LDL(-) uptake by primary macrophages and the progression of atherosclerotic lesions in Ldlr -/- mice. The engineered and optimized scFv-anti-LDL(-)-MCMN-Zn nanoformulation is internalized by human and murine macrophages in vitro by different endocytosis mechanisms. Moreover, macrophages exhibited lower LDL(-) uptake and reduced mRNA and protein levels of IL1B and MCP1 induced by LDL(-) when treated with this new nanoformulation. In a mouse model of atherosclerosis employing Ldlr -/- mice, intravenous administration of scFv-anti-LDL(-)-MCMN-Zn nanoformulation inhibited atherosclerosis progression without affecting vascular permeability or inducing leukocytes-endothelium interactions. Together, these findings suggest that a scFv-anti-LDL(-)-MCMN-Zn nanoformulation holds promise to be used in future preventive and therapeutic strategies for atherosclerosis.

Inflammasome Activation in Human Macrophages Induced by a LDL (-) Mimetic Peptide.

The inflammasome is responsible for maturation of interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) contributing to the inflammatory process in atherosclerosis. It is shown here that an electronegative low-density lipoprotein [LDL (-)] apoB-100 mimetic peptide can activate the transcriptional and posttranslational signs needed for complete inflammasome activation. This peptide, named p2C7, can activate the Toll-like receptor 4 (TLR4) that induces NF-κB activation and the transcription of inflammasome components. After blocking TLR4 with a neutralizing antibody, inflammasome component (NLRP3, CASP1, and ASC) and IL1b and IL18 gene downregulation occurred in human-derived macrophages stimulated with p2C7 or LDL (-). Moreover, the posttranslational signal was activated by the interaction between p2C7 and the lectin-type oxidized LDL receptor 1 (LOX-1), as demonstrated by the induction of caspase-1 cleavage in macrophages. The blockage of either TLR4 or LOX-1 decreased IL-1ß and IL-18 secretion by human-derived macrophages as both pathways are necessary for complete inflammasome activation. These findings suggest a mechanism by which macrophages transduce the pro-inflammatory signal provided by LDL (-) ApoB-100 and its mimetic peptides to activate the inflammasome protein complex what may be relevant for the inflammatory process in atherosclerosis.

Alpha-tocopherol supplementation decreases electronegative low-density lipoprotein concentration [LDL(-)] in haemodialysis patients.

BACKGROUND: Oxidative stress is a significant contributor to cardiovascular diseases (CVD) in haemodialysis (HD) patients, predisposing to the generation of oxidized low-density lipoprotein (oxLDL) or electronegatively charged LDL subfraction. Antioxidant therapy such as alpha-tocopherol acts as a scavenger of lipid peroxyl radicals attenuating the oxidative stress, which decreases the formation of oxLDL. The present study was designed to investigate the influence of the alpha-tocopherol supplementation on the concentration of electronegative low-density lipoprotein [LDL(-)], a minimally oxidized LDL, which we have previously described to be high in HD patients. METHODS: Blood samples were collected before and after 120 days of supplementation by alpha-tocopherol (400 UI/day) in 19 stable HD patients (50 +/- 7.8 years; 9 males). The concentrations of LDL(-) in blood plasma [using an anti-LDL- human monoclonal antibody (mAb)] and the anti-LDL(-) IgG auto-antibodies were determined by ELISA. Calculation of body mass index (BMI) and measurements of waist circumference (WC), triceps skin folds (TSF) and arm muscle area (AMA) were performed. RESULTS: The plasma alpha-tocopherol levels increased from 7.9 microM (0.32-18.4) to 14.2 microM (1.22-23.8) after the supplementation (P = 0.02). The mean concentration of LDL(-) was reduced from 570.9 microg/mL (225.6-1241.0) to 169.1 microg/mL (63.6-621.1) (P < 0.001). The anti-LDL(-) IgG auto-antibodies did not change significantly after the supplementation. The alpha-tocopherol supplementation also reduced the total cholesterol and LDL-C levels in these patients, from 176 +/- 42.3 mg/dL to 120 +/- 35.7 mg/dL (P < 0.05) and 115.5 +/- 21.4 mg/dL to 98.5 +/- 23.01 mg/dL (P < 0.001), respectively. CONCLUSION: The oral administration of alpha-tocopherol in HD patients resulted in a significant decrease in the LDL(-), total cholesterol and LDL-C levels. This effect may favour a reduction in cardiovascular risk in these patients, but a larger study is required to confirm an effect in this clinical setting.

Proinflammatory Action of a New Electronegative Low-Density Lipoprotein Epitope.

The electronegative low-density lipoprotein, LDL (-), is an endogenously modified LDL subfraction with cytotoxic and proinflammatory actions on endothelial cells, monocytes, and macrophages contributing to the progression of atherosclerosis. In this study, epitopes of LDL (-) were mapped using a phage display library of peptides and monoclonal antibodies reactive to this modified lipoprotein. Two different peptide libraries (X6 and CX8C for 6- and 8-amino acid-long peptides, respectively) were used in the mapping. Among all tested peptides, two circular peptides, P1A3 and P2C7, were selected based on their high affinities for the monoclonal antibodies. Small-angle X-ray scattering analysis confirmed their structures as circular rings. P1A3 or P2C7 were quickly internalized by bone marrow-derived murine macrophages as shown by confocal microscopy. P2C7 increased the expression of TNFα, IL-1 ß and iNOS as well as the secretion of TNFα, CCL2, and nitric oxide by murine macrophages, similar to the responses induced by LDL (-), although less intense. In contrast, P1A3 did not show pro-inflammatory effects. We identified a mimetic epitope associated with LDL (-), the P2C7 circular peptide, that activates macrophages. Our data suggest that this conformational epitope represents an important danger-associated molecular pattern of LDL (-) that triggers proinflammatory responses.

One-Year Conservative Care Using Sodium Bicarbonate Supplementation Is Associated with a Decrease in Electronegative LDL in Chronic Kidney Disease Patients: A Pilot Study.

BACKGROUND: Chronic kidney disease (CKD) patients develop metabolic acidosis when approaching stages 3 and 4, a period in which accelerated atherogenesis may ensue. Studies in vitro show that low pH may increase low-density lipoprotein (LDL) oxidation, suggesting a role for chronic metabolic acidosis in atherosclerosis. The present study attempted to evaluate the effects of conservative care using oral sodium bicarbonate (NaHCO3) supplementation on the electronegative LDL [LDL(-)], a minimally oxidized LDL, plasma levels in CKD patients. METHODS: Thirty-one CKD patients were followed by a multidisciplinary team during 15 months of care in which 1.0 mmol/kg/day oral NaHCO3 supplementation was first given in the third month. Blood samples were collected 3 months before the initiation of oral NaHCO3 supplementation (T1), at the time of the beginning of supplementation (T2), and thereafter, each 4 months (T3, T4 and T5) until month 15 of care. Blood parameters and LDL(-) were measured from these collections. RESULTS: After 12 months of conservative care, creatinine clearance (MDRD) was kept stable, and serum bicarbonate (HCO3-) increased from 20.5 ± 2.9 to 22.6 ± 1.1 mM (p < 0.003). LDL(-) plasma levels declined from 4.5 ± 3.3 to 2.1 ± 0.9 U/L (p < 0.007) after reaching mean serum HCO3- levels of 22.6 ± 1.1 mM. CONCLUSIONS: Conservative care using oral NaHCO3 supplementation was able to stabilize renal function and decrease serum levels of LDL(-), a modified proatherogenic lipoprotein, only when mean serum HCO3- levels approached 22 mM. This study constitutes evidence that alkali therapy, in addition to its beneficial effect on renal disease progression, might serve as a preventive strategy to attenuate atherogenesis in CKD patients.

Identification of mildly oxidized low-density lipoprotein (electronegative LDL) and its auto-antibodies IgG in children and adolescents hypercholesterolemic offsprings.

BACKGROUND: Oxidative modification of low-density lipoproteins (LDL) is an essential step in atherogenesis, generating minimally oxidized LDL, also called electronegative LDL [LDL(-)], which has chemotactic, cytotoxic and immunogenic properties. METHODS AND RESULTS: Serum LDL(-) and anti-LDL(-) auto-antibodies (IgG) were evaluated in 28 children and adolescents with familial hypercholesterolemia (FH) antecedents, with or without early coronary artery disease in first-degree relatives (eCAD), hypercholesterolemic (hc) or normocholesterolemic (nc) versus a control group of normocholesterolemic children without pathologic antecedents (C). ELISA method was used for detection of LDL(-) and anti-LDL(-) IgG. LDL(-) serum levels did not differ among the four groups (FH-eCAD-hc 41.4 +/- 24.9 microg/dl; FH-hc 38.3 +/- 11.2 microg/dl; FH-nc 47.3 +/- 17.0 microg/dl and C 44.2 +/- 28.8 microg/dl, p = 0.659). However, IgG anti-LDL(-) auto-antibodies were significantly higher in the control group in comparison to the FH groups with or without eCAD, independent of hypercholesterolemia or normocholesterolemia (FH-eCAD-hc 0.825 +/- 0.289 microg/dl; FH-hc 0.667 +/- 0.307 microg/dl; FH-nc 0.763 +/- 0.204 microg/dl and C 1.105 +/- 0.233 microg/dl, p = 0.006). When the auto-antibodies of groups with FH, with or without eCAD and with or without hypercholesterolemia were compared, no differences were found (p = 0.509). CONCLUSION: These results showed that FH and/or eCAD children and adolescents have lower titers of auto-antibodies anti-LDL(-) than children from normal families, independent of serum LDL-cholesterol or serum LDL(-).

Effects of soy germ isoflavones and hormone therapy on nitric oxide derivatives, low-density lipoprotein oxidation, and vascular reactivity in hypercholesterolemic postmenopausal women.

OBJECTIVE: To evaluate the effects of soy germ isoflavones and hormone therapy on vascular reactivity, the formation of nitric oxide derivatives, and lipid peroxidation in hypercholesterolemic postmenopausal women. DESIGN: Women were treated with soy germ, 17beta-estradiol or 17beta-estradiol + noretisterone acetate for 3 months after taking placebo for 1 month. The plasma concentrations of nitrite + nitrate and S-nitrosothiols were evaluated by gaseous phase chemiluminescence; nitrotyrosine, electronegative low-density lipoprotein, and estradiol levels were determined by enzyme-linked immunosorbent assay; cholesterol oxides and isoflavones were determined by gas chromatography and high-performance liquid chromatography, respectively. Vascular reactivity was analyzed by high-resolution ultrasonography. RESULTS: Soy germ isoflavones and hormone therapy induced a decrease in nitrite + nitrate, electronegative low-density lipoprotein, and cholesterol oxides, as well as an increase in S-nitrosothiols. Soy germ isoflavones lowered electronegative low-density lipoprotein, and cholesterol oxides more efficiently than did hormone therapy. Only soy isoflavones inhibited nitrotyrosine formation. A significant improvement of vascular reactivity was only seen in women treated with 17beta-estradiol. CONCLUSIONS: The soy germ isoflavones and 17beta-estradiol, alone or associated with noretisterone acetate, in the doses and forms used here, have similar effects on the bioavailability of nitric oxide. Soy germ treatment inhibited lipid peroxidation more effectively than hormone therapy.

A nanoformulation containing a scFv reactive to electronegative LDL inhibits atherosclerosis in LDL receptor knockout mice.

Atherosclerosis is a chronic inflammatory disease responsible for the majority of cases of myocardial infarction and ischemic stroke. The electronegative low-density lipoprotein, a modified subfraction of native LDL, is pro-inflammatory and plays an important role in atherogenesis. To investigate the effects of a nanoformulation (scFv anti-LDL(-)-MCMN-Zn) containing a scFv reactive to LDL(-) on the inhibition of atherosclerosis, its toxicity was evaluated in vitro and in vivo and further it was also administered weekly to LDL receptor knockout mice. The scFv anti-LDL(-)-MCMN-Zn nanoformulation did not induce cell death in RAW 264.7 macrophages and HUVECs. The 5mg/kg dose of scFv anti-LDL(-)-MCMN-Zn did not cause any typical signs of toxicity and it was chosen for the evaluation of its atheroprotective effect in Ldlr(-/-) mice. This nanoformulation significantly decreased the atherosclerotic lesion area at the aortic sinus, compared with that in untreated mice. In addition, the Il1b mRNA expression and CD14 protein expression were downregulated in the atherosclerotic lesions at the aortic arch of Ldlr(-/-) mice treated with scFv anti-LDL(-)-MCMN-Zn. Thus, the scFv anti-LDL(-)-MCMN-Zn nanoformulation inhibited the progression of atherosclerotic lesions, indicating its potential use in a future therapeutic strategy for atherosclerosis.

Probiotic Soy Product Supplemented with Isoflavones Improves the Lipid Profile of Moderately Hypercholesterolemic Men: A Randomized Controlled Trial.

BACKGROUND: Cardiovascular disease is the leading cause of worldwide morbidity and mortality. Several studies have demonstrated that specific probiotics affect the host's metabolism and may influence the cardiovascular disease risk. OBJECTIVES: The aim of this study was to investigate the influence of an isoflavone-supplemented soy product fermented with Enterococcus faecium CRL 183 and Lactobacillus helveticus 416 on cardiovascular risk markers in moderately hypercholesterolemic subjects. DESIGN: Randomized placebo-controlled double-blind trial Setting: São Paulo State University in Araraquara, SP, Brazil. PARTICIPANTS: 49 male healthy men with total cholesterol (TC) >5.17 mmol/L and <6.21 mmol/L Intervention: The volunteers have consumed 200 mL of the probiotic soy product (group SP-10(10) CFU/day), isoflavone-supplemented probiotic soy product (group ISP-probiotic plus 50 mg of total isoflavones/100 g) or unfermented soy product (group USP-placebo) for 42 days in a randomized, double-blind study. MAIN OUTCOME MEASURES: Lipid profile and additional cardiovascular biomarkers were analyzed on days 0, 30 and 42. Urine samples (24 h) were collected at baseline and at the end of the experiment so as to determine the isoflavones profile. RESULTS: After 42 days, the ISP consumption led to improved total cholesterol, non-HDL-C (LDL + IDL + VLDL cholesterol fractions) and electronegative LDL concentrations (reduction of 13.8%, 14.7% and 24.2%, respectively, p < 0.05). The ISP and SP have prevented the reduction of HDL-C level after 42 days. The C-reactive protein and fibrinogen levels were not improved. The equol production by the ISP group subjects was inversely correlated with electronegative LDL concentration. CONCLUSIONS: The results suggest that a regular consumption of this probiotic soy product, supplemented with isoflavones, could contribute to reducing the risk of cardiovascular diseases in moderately hypercholesterolemic men, through the an improvement in lipid profile and antioxidant properties.

AMP-Activated Protein Kinase Interacts with the Peroxisome Proliferator-Activated Receptor Delta to Induce Genes Affecting Fatty Acid Oxidation in Human Macrophages.

AMP-activated protein kinase (AMPK) maintains energy homeostasis by suppressing cellular ATP-consuming processes and activating catabolic, ATP-producing pathways such as fatty acid oxidation (FAO). The transcription factor peroxisome proliferator-activated receptor δ (PPARδ) also affects fatty acid metabolism, stimulating the expression of genes involved in FAO. To question the interplay of AMPK and PPARδ in human macrophages we transduced primary human macrophages with lentiviral particles encoding for the constitutively active AMPKα1 catalytic subunit, followed by microarray expression analysis after treatment with the PPARδ agonist GW501516. Microarray analysis showed that co-activation of AMPK and PPARδ increased expression of FAO genes, which were validated by quantitative PCR. Induction of these FAO-associated genes was also observed upon infecting macrophages with an adenovirus coding for AMPKγ1 regulatory subunit carrying an activating R70Q mutation. The pharmacological AMPK activator A-769662 increased expression of several FAO genes in a PPARδ- and AMPK-dependent manner. Although GW501516 significantly increased FAO and reduced the triglyceride amount in very low density lipoproteins (VLDL)-loaded foam cells, AMPK activation failed to potentiate this effect, suggesting that increased expression of fatty acid catabolic genes alone may be not sufficient to prevent macrophage lipid overload.

Predictive Potential of Twenty-Two Biochemical Biomarkers for Coronary Artery Disease in Type 2 Diabetes Mellitus.

We investigated the potential of a panel of 22 biomarkers to predict the presence of coronary artery disease (CAD) in type 2 diabetes mellitus (DM2) patients. The study enrolled 96 DM2 patients with (n = 75) and without (n = 21) evidence of CAD. We assessed a biochemical profile that included 22 biomarkers: total cholesterol, LDL, HDL, LDL/HDL, triglycerides, glucose, glycated hemoglobin, fructosamine, homocysteine, cysteine, methionine, reduced glutathione, oxidized glutathione, reduced glutathione/oxidized glutathione, L-arginine, asymmetric dimethyl-L-arginine, symmetric dimethyl-L-arginine, asymmetric dimethyl-L-arginine/L-arginine, nitrate plus nitrite, S-nitrosothiols, nitrotyrosine, and n-acetyl-ß-glucosaminidase. Prediction models were built using logistic regression models. We found that eight biomarkers (methionine, nitratate plus nitrite, n-acetyl-ß-glucosaminidase, BMI, LDL, HDL, reduced glutathione, and L-arginine/asymmetric dimethyl-L-arginine) along with gender and BMI were significantly associated with the odds of CAD in DM2. These preliminary findings support the notion that emerging biochemical markers might be used for CAD prediction in patients with DM2. Our findings warrant further investigation with large, well-designed studies.

The hypolipidemic and pleiotropic effects of rosuvastatin are not enhanced by its association with zinc and selenium supplementation in coronary artery disease patients: a double blind randomized controlled study.

OBJECTIVE: Statins treatment may modify the levels of zinc and selenium, minerals that can improve vascular function and reduce oxidative damage and inflammation in atherosclerotic patients. This study aimed to evaluate the effects of rosuvastatin, alone or associated with zinc and selenium supplementation, on lipid profile, antioxidant enzymes and mineral status in coronary artery disease patients. MATERIAL AND METHODS: A double-blind randomized clinical trial was performed in which patients (n = 76) were treated with 10 mg rosuvastatin over 4 months associated or not with zinc (30 mg/d) and selenium (150 µg/d) supplementation. The following parameters were analyzed before and after the intervention: anthropometric measurements, lipid profile, high sensitivity C-reactive protein (hs-CRP), electronegative low density lipoprotein (LDL(-)) concentrations, activities of glutathione peroxidase (GPx), superoxide dismutase (SOD), zinc and selenium concentrations in blood plasma and erythocytes. Significance was determined using an α of 5% (two-tailed). RESULTS: We found that rosuvastatin therapy was efficient in reducing total cholesterol, LDL-cholesterol, non-HDL cholesterol, triglycerides, and hs-CRP independently of mineral supplementation. Neither treatment was associated with significant changes in LDL(-). Similarly, the antioxidant enzymes GPx and SOD activity were unchanged by treatments. Neither treatment was associated with significant differences in concentrations of zinc or selenium in blood plasma and erythocytes of studied groups. CONCLUSION: Rosuvastatin treatment did not affect zinc and selenium levels in coronary artery disease patients. The zinc and selenium supplementation at doses used in this study did not change lipid profile or SOD and GPx activity in patients receiving rosuvastatin. Further studies should be focused on testing alternative doses and supplements in different populations to contribute for a consensus on the ideal choice of antioxidants to be used as possible complementary therapies in atherosclerotic patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT01547377.

Improved nonclinical pharmacokinetics and biodistribution of a new PPAR pan-agonist and COX inhibitor in nanocapsule formulation.

We report the in vitro release profile and comparative pharmacokinetics and biodistribution of a new peroxisome proliferator-activated receptor-γ agonist and cyclooxygenase inhibitor (Lyso-7) free or associated to poly(D,L-lactic acid) nanocapsules (NC) after intravenous administration in mice. Lyso-7 pertains to the class of insulin-sensitizing agents that shows potential beneficial effects in diabetes therapy. Monodispersed Lyso-7 NC with a mean diameter of 273 nm with high encapsulation efficiency (83%) were obtained. Lyso-7 dissolution rate was reduced (2.6-fold) upon loading in NC. The pharmacokinetic parameters were determined using a non-compartmental approach. In comparison with Lyso-7 in solution, the plasma-AUC increased 14-fold, the mean residence time 2.6-fold and the mean half-life (t1/2) 1.5-fold for Lyso-7-NC; the Lyso-7 plasma clearance, distribution volume and elimination rate were reduced 13, 10 and 1.4 fold, respectively, which indicates higher retention of encapsulated Lyso-7 in the blood compartment. Upon association with NC, organ exposure to Lyso-7 was higher in the heart (3.6-fold), lung (2.8-fold), spleen (2.3-fold), kidney (2-fold) and liver (1.8-fold) compared to Lyso-7 in solution. The analysis of whole data clearly indicates that body exposure to Lyso-7 was enhanced and the general toxicity reduced upon nanoencapsulation, allowing further evaluation of Lyso-7 in nonclinical and clinical studies.

Progesterone abolishes estrogen and/or atorvastatin endothelium dependent vasodilatory effects.

UNLABELLED: This double blind randomized placebo controlled study assessed the effects of atorvastatin, estradiol and norethisterone, isolated and in combination, on the lipid profile and on vascular reactivity, in post-menopausal women with hypercholesterolemia and arterial hypertension. Ninety-four women aged 50-65 were selected. All have received dietary counseling (4 weeks), placebo (4 weeks), and drug therapy (12 weeks): 17-beta estradiol 2mg/day (E) (n=17); E + norethisterone acetate 1mg/day (P) (n=18); Atorvastatin 10mg/day (A) (n=20); E + A (n=21) and E + P + A (n=18). All treatment modalities have significantly reduced total cholesterol (TC) (E=8.8%, E + P=10.1%, A=27.9%, A + E=29.4% and E + P + A=35.7%) and LDL-cholesterol (LDL-c) levels (E + P + A=46.6%, E + A=45.9%, A=40.2%, E=20.3%, and E + P=12.1%). As concerns HDL-cholesterol (HDL-c), Groups E and E + A had increases of 15.5% and 13.1%, respectively. The addition of a progesterone compound reduced its concentration (Group E + P=-9.1%, and Group E + P + A=-9.5%). By random, approximately half of the patients in each group were designated to the endothelial function evaluation (brachial artery ultrasound). We observed that in Group A (n=10), in Group E (n=10) and with the association (Group E + A) (n=7), there was a significant increase in the flow-mediated vasodilatation as compared to basal measurements. The addition of a progestin has annulled these benefits. CONCLUSIONS: Atorvastatin has promoted more beneficial effects on TC and LDL-c, whereas estradiol was responsible for an increase in HDL-c. The addition of a progesterone derivative abolished these benefits. Atorvastatin, estradiol or both together improved endothelial function, an effect suppressed by the addition of norethisterone.

Effect of N-acetyl-L-cysteine on lymphocyte apoptosis, lymphocyte viability, TNF-alpha and IL-8 in HIV-infected patients undergoing anti-retroviral treatment.

N-acetyl-L-cysteine (NAC) has been proposed as an additional therapeutic agent for AIDS patients because it reduces human immunodeficiency virus type 1 (HIV-1) replication in stimulated CD4+ lymphocytes, and it ameliorates immunological reactivity. In a randomized, 180-day, double-blind, placebo-controlled trial performed with HIV-infected patients classified as A2 and A3 according to the criteria of the Center for Disease Control and Prevention, we investigated the effects of oral administration of NAC on HIV-infected patients undergoing their first anti-retroviral therapy; viral load, CD4+ lymphocyte, lymphocyte viability and apoptosis, and TNF-alpha and IL-8 levels were determined. Sixteen patients who received anti-retroviral therapy plus a placebo formed the control group and the study group consisted of 14 patients who received anti-retroviral therapy and NAC supplementation. A significant decrease was seen in viral load, TNF-alpha and IL-8 levels, and lymphocyte apoptosis, and a significant increase was found in levels of CD4+ lymphocytes and lymphocyte viability in both groups after anti-retroviral treatment, but no measurable benefits of anti-retroviral therapy plus NAC oral supplementation (600 mg/day) were found in relation to anti-retroviral therapy alone, and the baseline levels of cysteine and glutathione in plasma were not recovered by this treatment. In conclusion, the daily doses of NAC necessary for the total recuperation of plasma cysteine and glutathione levels in HIV-infected patients and the additional benefits following the supplementation of NAC in patients submitted to anti-retroviral therapy, need to be studied further.