Polyphenols and Triterpenes Combination in an In Vitro Model of Cardiac Damage: Protective Effects.

Olive products contain high levels of monounsaturated fatty acids as well as other minor components such as triterpenic alcohols and other pentacyclic triterpenes, which together form the main triterpenes of virgin olive oil. Olive fruits and leaves contain significant amounts of hydrophilic and lipophilic bioactives including flavones, phenolic acids and phenolic alcohols, amongst others. Several studies have shown the benefits of these substances on the cardiovascular system. Regardless, little is known about the specific combination of bioactive compounds in cardiovascular health. Thus, we aimed to test the combination of a triterpenes (TT70) and a polyphenols (HT60) olive oil bioactive extract in H9c2 cells under stress conditions: LPS and H2O2 stimulation. To evaluate the effectiveness of the combination, we measured cell viability, superoxide production and protein expression of caspase 3, eNOS, peNOS, TNF-α and Il-6. Overall, cells stimulated with LPS or H2O2 and co-incubated with the combination of triterpenes and polyphenols had increased cell survival, lower levels of superoxide anion, lower protein expression of eNOS and higher expression of peNOS, increased protein expression of SOD-1 and lower protein expression of TNF-α and Il-6. The specific combination of HT60+TT70 is of great interest for further study as a possible treatment for cardiovascular damage.

Long-term exposure to nanoplastics alters molecular and functional traits related to the carcinogenic process.

Micro/nanoplastics (MNPLs) are considered emergent pollutants widely spread over all environmental compartments. Although their potential biological effects are being intensively evaluated, many doubts remain about their potential health effects in humans. One of the most underdeveloped fields is the determination of the potential tumorigenic risk of MNPLs exposure. To shed light on this topic, we have designed a wide battery of different hallmarks of cancer applied to prone-to-transformed progress MEF cells exposed to polystyrene nanoplastics (PSNPLs) in the long term (6 months). Interestingly, most of the evaluated hallmarks of cancer are exacerbated after exposure, independently if they are associated with an early tumoral phenotype (changes in stress-related genes, or microRNA deregulation), advanced tumoral phenotype (growing independently of anchorage ability, and migration capacity), or an aggressive tumoral phenotype (invasion potential, changes in pluripotency markers, and ability to grow to form tumorspheres). This set of obtained data constitutes a relevant warning on the potential carcinogenic risk associated with long-term exposures to MNPLs, specifically that induced by the PSNPLs evaluated in this study.

CRISPR/Cas9-Directed Gene Trap Constitutes a Selection System for Corrected BCR/ABL Leukemic Cells in CML.

Chronic myeloid leukaemia (CML) is a haematological neoplasm driven by the BCR/ABL fusion oncogene. The monogenic aspect of the disease and the feasibility of ex vivo therapies in haematological disorders make CML an excellent candidate for gene therapy strategies. The ability to abolish any coding sequence by CRISPR-Cas9 nucleases offers a powerful therapeutic opportunity to CML patients. However, a definitive cure can only be achieved when only CRISPR-edited cells are selected. A gene-trapping approach combined with CRISPR technology would be an ideal approach to ensure this. Here, we developed a CRISPR-Trap strategy that efficiently inserts a donor gene trap (SA-CMV-Venus) cassette into the BCR/ABL-specific fusion point in the CML K562 human cell line. The trapping cassette interrupts the oncogene coding sequence and expresses a reporter gene that enables the selection of edited cells. Quantitative mRNA expression analyses showed significantly higher level of expression of the BCR/Venus allele coupled with a drastically lower level of BCR/ABL expression in Venus+ cell fractions. Functional in vitro experiments showed cell proliferation arrest and apoptosis in selected Venus+ cells. Finally, xenograft experiments with the selected Venus+ cells showed a large reduction in tumour growth, thereby demonstrating a therapeutic benefit in vivo. This study represents proof of concept for the therapeutic potential of a CRISPR-Trap system as a novel strategy for gene elimination in haematological neoplasms.

MicroRNAs as a Suitable Biomarker to Detect the Effects of Long-Term Exposures to Nanomaterials. Studies on TiO2NP and MWCNT.

The presence of nanomaterials (NMs) in the environment may represent a serious risk to human health, especially in a scenario of chronic exposure. To evaluate the potential relationship between NM-induced epigenetic alterations and carcinogenesis, the present study analyzed a panel of 33 miRNAs related to the cell transformation process in BEAS-2B cells transformed by TiO2NP and long-term MWCNT exposure. Our battery revealed a large impact on miRNA expression profiling in cells exposed to both NMs. From this analysis, a small set of five miRNAs (miR-23a, miR-25, miR-96, miR-210, and miR-502) were identified as informative biomarkers of the transforming effects induced by NM exposures. The usefulness of this reduced miRNA battery was further validated in other previously generated transformed cell systems by long-term exposure to other NMs (CoNP, ZnONP, MSiNP, and CeO2NP). Interestingly, the five selected miRNAs were consistently overexpressed in all cell lines and NMs tested. These results confirm the suitability of the proposed set of mRNAs to identify the potential transforming ability of NMs. Particular attention should be paid to the epigenome and especially to miRNAs for hazard assessment of NMs, as wells as for the study of the underlying mechanisms of action.

Nanoceria, alone or in combination with cigarette-smoke condensate, induce transforming and epigenetic cancer-like features in vitro.

Aim: To detect cell transformation effects of nanoceria after long-term exposure (up to 6 weeks) and to determine their potential interactions with cigarette smoke condensate, as a model of environmental carcinogenic pollutant. Materials & methods: Human bronchial epithelial BEAS-2 cells were used to determine transformation effects (invasion and tumorspheres induction), as well as changes in the expression of a battery of miRNAs related to the carcinogenesis process. Results: Nanoceria- and co-exposed cells exhibit cell transforming potential, with significantly increased invasion and tumorsphere formation abilities. Likewise, these exposures produced a high impact on the battery of miRNAs used. Conclusion: Nanoceria exposure induces cell-transformation and shows a positive interaction with the cell-transforming effects of cigarette smoke condensate. Besides, cerium dioxide nanoparticles and the co-exposure produced potential toxicity at the transcriptome level, which is related to tumorigenesis.

Effect of Pectin on the Expression of Proteins Associated with Mitochondrial Biogenesis and Cell Senescence in HT29-Human Colorectal Adenocarcinoma Cells.

Mitochondria dynamic is regulated by different proteins, maintaining a balance between fission and fusion. An imbalance towards mitochondrial fission has been associated with tumor cell proliferation. The aim of this study was to analyze whether pectin modifies the viability of human colon cancer cells and the expression of proteins involved in mitochondrial fusion and fission. The human colon carcinoma cell line HT29 cells was growth in 10% fetal bovine serum in the absence and presence of pectin. Pectin reduced HT29 cell viability in a concentration-dependent manner, reaching a plateau at 150~300 µmol/L pectin. The presence of 200 µmol/L pectin reduced the expression of dynamin-related protein-1 and increased expression of the mitochondrial fusion-associated proteins mitofusin-1 and 2. Expression of cyclin B1, a protein involved in G2/M transition, was found decreased in pectin-incubated HT29 cells. Moreover, expression of p53 protein, the amount of p53 in the nucleous and ß-galactosidase activity, which are all biomarkers for cellular senescence, were significantly higher in pectin-incubated HT29 cells than in HT29 cells incubated without pectin. Expression of the protein B-cell lymphoma 2 (Bcl-2) homologous antagonist/killer was increased in response to incubation with pectin. However, incubation with pectin did not affect expression of Bcl-2-associated X protein or Bcl-2, or the caspase-3 activity. Overall, we concluded that pectin reduces the viability of human HT29 colon cancer cells, which is accompanied with a shift in the expression of proteins associated with mitochondrial dynamics towards mitochondrial fusion. Moreover, incubation with pectin favors cellular senescence over apoptosis in HT29 cells.

Supplementation with an insoluble fiber obtained from carob pod (Ceratonia siliqua L.) rich in polyphenols prevents dyslipidemia in rabbits through SIRT1/PGC-1α pathway.

PURPOSE: To investigate the mechanism implicated in the effect of an insoluble fiber (obtained from carob pod) rich in polyphenols (IFCP) in lipid metabolism in the liver. METHODS: Male New Zealand rabbits were fed with the following diets for 8 weeks: control diet (CT group), dyslipidemic diet supplemented with 0.5% cholesterol + 14% coconut oil (DL group) and dyslipidemic diet containing 0.5% cholesterol + 14% coconut oil plus 3% IFCP (DL + IFCP group). RESULTS: Dyslipidemic diet with IFCP was able to reduce development of mixed dyslipidemia, liver relative weight and collagen I protein expression compared to DL rabbits. Analyses of the main enzymes implicated in cholesterol and triglycerides metabolism revealed that IFCP increased hepatic concentration of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and cytochrome P450, family 7, subfamily a, polypeptide 1C (CYP7A1) (82.34, 114.42%, respectively) as well as protein expression of LDL receptor (42.48%) in DL rabbits. Importantly, IFCP also increased hepatic lipase (HL) levels (91.43%) and decreased glycerol phosphate acyltransferase (GPAT) and sterol regulatory element-binding protein 1C (SREBP1c) liver expression levels (20.38 and 41.20%, respectively). Finally, sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1α) hepatic expression increased in DL + IFCP group compared with DL (159.81 and 48.00%, respectively). CONCLUSIONS: These findings show that IFCP is able to abrogate the deleterious effects of hepatic dyslipidemia by modulating SIRT1 and PGC-1α pathways.

Low Phytanic Acid-Concentrated DHA Prevents Cognitive Deficit and Regulates Alzheimer Disease Mediators in an ApoE-/- Mice Experimental Model.

Alzheimer's disease (AD) is the main cause of dementia and cognitive impairment. It has been associated with a significant diminution of omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) levels in the brain. Clinical trials with DHA as a treatment in neurological diseases have shown inconsistent results. Previously, we reported that the presence of phytanic acid (PhA) in standard DHA compositions could be blunting DHA's beneficial effects. Therefore, we aimed to analyze the effects of a low PhA-concentrated DHA and a standard PhA-concentrated DHA in Apolipoprotein E knockout (ApoE-/-) mice. Behavioral tests and protein expression of pro-inflammatory, pro-oxidant, antioxidant factors, and AD-related mediators were evaluated. Low PhA-concentrated DHA decreased Aß, ß-amyloid precursor protein (APP), p-tau, Ca2+/calmodulin-dependent protein kinase II (CAMKII), caspase 3, and catalase, and increased brain derived neurotrophic factor (BDNF) when compared to standard PhA-concentrated DHA. Low PhA-concentrated DHA decreased interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α) protein expression in ApoE-/- mice when compared to standard PhA-concentrated DHA. No significant differences were found in p22phox, inducible nitric oxide synthase (iNOS), glutathione peroxidase (GPx), superoxide dismutase 1 (SOD-1), and tau protein expression. The positive actions of a low PhA-concentrated DHA were functionally reflected by improving the cognitive deficit in the AD experimental model. Therefore, reduction of PhA content in DHA compositions could highlight a novel pathway for the neurodegeneration processes related to AD.

Aldosterone Induces Renal Fibrosis and Inflammatory M1-Macrophage Subtype via Mineralocorticoid Receptor in Rats.

We aimed to evaluate macrophages heterogeneity and structural, functional and inflammatory alterations in rat kidney by aldosterone + salt administration. The effects of treatment with spironolactone on above parameters were also analyzed. Male Wistar rats received aldosterone (1 mgkg-1d-1) + 1% NaCl for 3 weeks. Half of the animals were treated with spironolactone (200 mg kg-1d-1). Systolic and diastolic blood pressures were elevated (p<0.05) in aldosterone + salt-treated rats. Relative kidney weight, collagen content, fibronectin, macrophage infiltrate, CTGF, Col I, MMP2, TNF-α, CD68, Arg2, and SGK-1 were increased (p<0.05) in aldosterone + salt-treated rats, being reduced by spironolactone (p<0.05). Increased iNOS and IFN-γ mRNA gene expression (M1 macrophage markers) was observed in aldosterone + salt rats, whereas no significant differences were observed in IL-10 and gene ArgI mRNA expression or ED2 protein content (M2 macrophage markers). All the observed changes were blocked with spironolactone treatment. Macrophage depletion with liposomal clodronate reduced macrophage influx and inflammatory M1 markers (INF-γ or iNOS), whereas interstitial fibrosis was only partially reduced after this intervention, in aldosterone plus salt-treated rats. In conclusion, aldosterone + salt administration mediates inflammatory M1 macrophage phenotype and increased fibrosis throughout mineralocorticoid receptors activation.

Relevance of SGK1 in structural, functional and molecular alterations produced by aldosterone in heart.

Aldosterone regulates sodium (Na+) and potassium (K+) transports in epithelial cells. Besides, aldosterone participates in cardiac alterations associated with hypertension, heart failure, diabetes, and other pathological alterations. One of the main cardiac alterations induced by aldosterone is cardiac hypertrophy in which different mechanisms are involved such as increased cardiomyocyte, calcium concentration, oxidative stress, and inflammatory and fibrotic mediators stimulation. Many epidemiological studies have demonstrated that left ventricular hypertrophy is associated with significantly increased risk of heart failure and malignant arrhythmias. SGK1 is a member of the serine/threonine kinase gene family that plays an important role in the absorption of Na+ and water through the Na+ channel in the apical membrane of tubular epithelial cells. SGK1 has been related to fibrotic mediator increase such as connective tissue growth factor (CTGF) and transforming growth factor-ß (TGF-ß) as well as inflammatory [tumor necrosis factor-α (TNF-α) and interleukin (IL)-1ß] and oxidative (NADPH oxidase) species. It has been shown that aldosterone induces SGK1 gene expression not only in kidneys but also in the heart. Supporting the central role of SGK1 in cardiac alterations induced by aldosterone, treatment with the mineralocorticoid antagonist spironolactone is able to reduce the gene expression of SGK1 in aldosterone-treated rats. Taken together, data suggest the involvement of SGK1 in a complex intracellular signaling, involving fibrotic, inflammatory, and oxidative pathways, which lead to cardiac hypertrophy and fibrosis induced by aldosterone.

Carob pod insoluble fiber exerts anti-atherosclerotic effects in rabbits through sirtuin-1 and peroxisome proliferator-activated receptor-γ coactivator-1α.

The aim of this study was to evaluate the potential effects of an insoluble dietary fiber from carob pod (IFC) (1 g ⋅ kg(-1) ⋅ d(-1) in the diet) on alterations associated with atherosclerosis in rabbits with dyslipidemia. Male New Zealand rabbits (n = 30) were fed the following diets for 8 wk: 1) a control diet (SF412; Panlab) as a control group representing normal conditions; 2) a control supplemented with 0.5% cholesterol + 14% coconut oil (DL) (SF302; Panlab) for 8 wk as a dyslipidemic group; and 3) a control containing 0.5% cholesterol + 14% coconut oil plus IFC (1 g ⋅ kg(-1) ⋅ d(-1)) (DL+IFC) for 8 wk. IFC was administered in a pellet mixed with the DL diet. The DL-fed group developed mixed dyslipidemia and atherosclerotic lesions, which were associated with endothelial dysfunction, inflammation, and fibrosis. Furthermore, sirtuin-1 (SIRT1) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) protein expression in the aorta were reduced to 77% and 63% of the control group, respectively (P < 0.05), in these rabbits. Administration of IFC to DL-fed rabbits reduced the size of the aortic lesion significantly (DL, 15.2% and DL+IFC, 2.6%) and normalized acetylcholine-induced relaxation (maximal response: control, 89.3%; DL, 61.6%; DL+IFC, 87.1%; P < 0.05) and endothelial nitric oxide synthase expression (DL, 52% and DL+IFC, 104% of the control group). IFC administration to DL-fed rabbits also reduced cluster of differentiation 36 (DL, 148% and DL+IFC, 104% of the control group; P < 0.05), plasminogen activator inhibitor-1 (DL, 141% and DL+IFC, 107% of the control group), tumor necrosis factor-α (DL, 166% and DL+IFC, 120% of the control group), vascular cell adhesion molecule-1 (DL, 153% and DL+IFC, 110% of the control group), transforming growth factor-ß (DL, 173% and DL+IFC, 99% of the control group), and collagen I (DL, 157% and DL+IFC, 112% of the control group) in the aorta. These effects were accompanied by an enhancement of SIRT1 and PGC-1α (160% and 121% of the control group, respectively; P < 0.05) vascular expression. In summary, we demonstrated for the first time, to our knowledge, that administration of IFC reduces the development of atherosclerosis in rabbits. This effect seems to be related to an improvement in endothelial function and a reduction of inflammation and fibrosis, most probably as a consequence of the reduction of serum concentrations of cholesterol and triglycerides. Increased expression of aortic SIRT1 and PGC-1α could play an important role in the observed effects of IFC in rabbits with dyslipidemia.

Protective effect of a pomace olive oil concentrated in triterpenic acids in alterations related to hypertension in rats: mechanisms involved.

SCOPE: Despite the amount of information and research on the effects of virgin olive oil and its components in cardiovascular disease, little attention has been paid to the effects of pomace olive oil, an olive oil subproduct traditionally used in Spain. The aim of the present study was to evaluate the potential effects of a pomace olive oil concentrated in triterpenic acids (POCTA) on blood pressure, cardiac hemodynamics, and functional and molecular vascular alterations associated with hypertension in spontaneously hypertensive rats (SHR). METHODS AND RESULTS: The study showed that POCTA attenuated the increase of blood pressure in SHR. This effect was associated with an improvement in endothelium-dependent relaxation, enhancement of vascular expression of endothelial nitric oxide synthase, and reduction of tumor necrosis factor alpha, transforming growth factor beta, and collagen I. Furthermore, POCTA improved cardiac hemodynamics (left ventricular systolic pressure and left ventricular end-diastolic pressure) and decreased relative kidney and lung weights. CONCLUSION: POCTA exerts antihypertensive effects together with vascular and hypertension target organ protection in SHR. Since interest in pomace olive oil has been low, the results of this study contribute to increasing awareness of its biological and nutritional values.

Spironolactone prevents alterations associated with cardiac hypertrophy produced by isoproterenol in rats: involvement of serum- and glucocorticoid-regulated kinase type 1.

Persistent ß-adrenergic receptor stimulation with isoproterenol is associated with cardiac hypertrophy as well as cardiac synthesis of angiotensin II. Serum- and glucocorticoid-regulated kinase type 1 (SGK-1) is a key mediator in structural, functional and molecular cardiac effects of aldosterone in rats. This study was designed to investigate the cardiac effects of the mineralocorticoid receptor antagonist spironolactone on the response to isoproterenol treatment in rats, as well as the involvement of the main mediator of cellular aldosterone action, SGK-1, in the heart. Male Wistar rats received isoproterenol (3 mg kg(-1) day(-1)) or vehicle for 15 days. Half of the animals in each group were simultaneously treated with spironolactone (200 mg kg(-1) day(-1)). Systolic and diastolic blood pressures were not significantly different among groups. Treatment with spironolactone normalized the increased left ventricular end-diastolic pressure observed in isoproterenol-treated rats. Isoproterenol treatment induced cardiac hypertrophy and increased collagen content, both of which were normalized by spironolactone treatment. The mRNA levels of transforming growth factor ß, connective tissue growth factor, matrix metalloprotease 2, matrix metalloprotease inhibitor 2, tumour necrosis factor α, interleukin 1ß, p22phox and xanthine dehydrogenase were increased (P < 0.05) in isoproterenol-treated rats, and this effect was prevented by spironolactone (P < 0.05). Spironolactone also reduced the elevated SGK-1 expression in isoproterenol-treated rats. The observed reduction of the principal mediator of aldosterone cellular actions, SGK-1, by spironolactone in hearts from isoproterenol-treated rats suggests a role of mineralocorticoids in the cardiac hypertrophy, fibrosis, inflammation, oxidation and diastolic dysfunction induced by isoproterenol treatment in rats.

Structural, functional, and molecular alterations produced by aldosterone plus salt in rat heart: association with enhanced serum and glucocorticoid-regulated kinase-1 expression.

We aimed to evaluate the structural, functional, inflammatory, and oxidative alterations, as well as serum and glucocorticoid-regulated kinase-1 (SGK-1) expression, produced in rat heart by aldosterone + salt administration. Fibrosis mediators such as connective tissue growth factor, matrix metalloproteinase 2, and tissue inhibitor of metalloproteinases 2 were also evaluated. Treatment with spironolactone was evaluated to prove mineralocorticoid mediation. Male Wistar rats received aldosterone (1 mg[middle dot]kg-1[middle dot]d-1) + 1% NaCl for 3 weeks. Half of the animals were treated with spironolactone (200 mg[middle dot]kg-1[middle dot]d-1). Systolic and diastolic blood pressures, left ventricle (LV) systolic pressure, and LV end-diastolic pressure were elevated (P < 0.05) in aldosterone + salt-treated rats. In aldosterone + salt-treated rats, -dP/dt decreased (P < 0.05), but +dP/dt was similar in all groups. Spironolactone normalized (P < 0.05) systolic blood pressure, diastolic blood pressure, LV systolic pressure, LV end-diastolic pressure, and -dP/dt. Relative heart weight, collagen content, messenger RNA expression of transforming growth factor beta, connective tissue growth factor, matrix metalloproteinase 2, tissue inhibitor of metalloproteinases 2, tumor necrosis factor alpha, interleukin-1[beta], p22phox, endothelial nitric oxide synhtase, and SGK-1 were increased (P < 0.05) in aldosterone + salt-treated rats, being reduced by spironolactone (P < 0.05). SGK-1 might be a key mediator in the structural, functional, and molecular cardiac alterations induced by aldosterone + salt in rats. All the observed changes and mediators are related with the activation of mineralocorticoid receptors.