Atorvastatin’s Therapeutic Potential in Atherosclerosis: Inhibiting TGF-β-Induced Proteoglycan Glycosaminoglycan Chain Elongation through ROS-ERK1/2-Smad2L Signaling Pathway Modulation in Vascular Smooth Muscle Cells

Objective: According to the response-to-retention hypothesis, the inception of atherosclerosis is attributed to thedeposition and retention of lipoprotein in the arterial intima, facilitated by altered proteoglycans with hyperelongatedglycosaminoglycan (GAG) chains. Recent studies have elucidated a signaling pathway whereby transforming growthfactor-β (TGF-β) promotes the expression of genes linked to proteoglycan GAG chain elongation (CHSY1 and CHST11)via reactive oxygen species (ROS) and the downstream phosphorylation of ERK1/2 and Smad2L. Atorvastatin is knownto exhibit pleiotropic effects, including antioxidant and anti-inflammatory. The purpose of the present research wasto ascertain the influence of atorvastatin on TGF-β-stimulated expression of CHST11 and CHSY1 and associatedsignaling pathways using an in vitro model.Materials and Methods: In this experimental study, vascular smooth muscle cells (VSMCs) were pre-incubatedwith atorvastatin (0.1-10 μM) prior to being stimulated with TGF-β (2 ng/ml). The experiment aimed to evaluate thephosphorylation levels of Smad2C, Smad2L, ERK1/2, the NOX p47phox subunit, ROS production, and the mRNAexpression of CHST11 and CHSY1.Results: Our research results indicated that atorvastatin inhibited TGF-β-stimulated CHSY1 and CHST11 mRNAexpression. Further experiments showed that atorvastatin diminished TGF-β-stimulated ROS production and weakenedTGF-β-stimulated phosphorylation of p47phox, ERK1/2, and Smad2L; however, we observed no effect on the TGF-β-Smad2C pathway.Conclusion: These data suggest that atorvastatin demonstrates anti-atherogenic properties through the modulationof the ROS-ERK1/2-Smad2L signaling pathway. This provides valuable insight into the potential mechanisms by whichatorvastatin exerts its pleiotropic effects against atherosclerosis

Reactive Oxygen Species and p38MAPK Have a Role in the Smad2 Linker Region Phosphorylation Induced by TGF-β

Background: Transforming growth factor-β (TGF-β) in addition to the C-terminal region can phosphorylate receptor-regulated Smads (R-Smads) in their linker region. The aim of the present study was to evaluate the role of signaling mediators such as NAD(P)H oxidases (reactive oxygen species [ROS] generators), ROS, and ROS-sensitive p38 mitogen-activated protein kinase (p38MAPK) in this signaling pathway in cultured human vascular smooth muscle cells (VSMCs). Methods: The present in vitro study was performed on human VSMCs. Proteins were detected by western blotting utilizing an anti-phospho-Smad2 (Ser245/250/255) rabbit polyclonal antibody and a horseradish peroxidase-labeled secondary antibody. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. The phospho-Smad2 linker region (pSmad2L) was detected in all the experimental groups: a control group (untreated group), a group treated with TGF-β (2 ng/mL), and a group treated with TGF-β plus different inhibitors. The data were normalized and presented as mean ± SEM. The statistical analyses were performed using SPSS, version 16.0, and the nonparametric Kruskal–Wallis test. A P value smaller than 0.05 was considered statistically significant. Results: The VSMCs treated with TGF-β (2 ng/mL) showed a time-dependent increase in the pSmad2L level. The highest level was observed at 15 minutes (P=0.03). The inhibitors of NAD(P) H oxidases (diphenyleneiodonium and apocynin) (P=0.04), ROS scavenger (N-acetylcysteine) (P=0.04), and p38MAPK inhibitor (SB-202190) (P=0.04) were able to reduce the increased level of the pSmad2L by TGF-β. Conclusion: Our results suggested that NAD(P)H oxidases played an important role in the Smad2L phosphorylation in the human VSMCs. Furthermore, our results confirmed that ROS and p38MAPK were involved in this signaling pathway. Thus, TGF-β via a ROS-dependent mechanism can transmit its signals to the pSmad2L

Effect of elaidic acid on ABCA1 expression in RAW 264.7 cells.

In recent years, Trans Fatty Acids have shown a strong correlation with cardiovascular disease. However, the mechanisms explaining their atherogenicity are still unclear. ABCA1, which is involved in the reverse cholesterol transport pathway, has been considered as a new therapeutic target for cardiovascular disease. In vitro studies of the effects of PPAR-γ on lipid homeostasis in macrophage cells suggested a role for PPAR-γ in the regulation of ABCA1-dependent cholesterol efflux to apoA-I pathway. Thus, in this study we examined the effect of elaidic acid (EA) as the most abundant TFA on expression of ABCA1 and PPAR-γ in RAW 264.7 mouse macrophage cell line. Accordingly, after determining appropriate concentrations of EA using MTT, RAW 264.7 cells were treated with different concentrations of EA, and at the end, gene expression was assayed by Real-Time PCR. Our results shown that the expression of ABCA1 decreased in the treated group in comparison with the control group by 1.7, 2.3, and 5.1 fold, after 12 h treatment for 0.5, 1, and 2 mM EA concentration respectively. In addition, after 24 h treatment with EA, the rate of decreasing ABCA1 expression was 2.1, 2.6, 5.7 fold, respectively (P < 0.01). However, EA had no significant effect on PPAR-γ mRNA expression. Therefore, it could be concluded that the atherogenic effect of EA may be mediated by reducing ABCA1 expression in RAW 264.7 cells; however, this reduction has not mediated through altering PPAR-γ expression

Curcumin as an Environmental Potent Antioxidant Decreases Risk of Arthrosclerosis

Background & Aims of the Study: Oxidative stress increases platelet-derived growth factor (PDGF) gene expression in endothelial cells that contributes to vascular dysfunction and atherosclerosis. Oxidative stress generates by dys-regulated redox balance between ROS producing systems and antioxidant systems. Also, Curcumin (Cur) as a main part of turmeric has anti- inflammatory, antioxidant, anticancer and antitumor effects. This study was conducted to test the Curcumin as an environmental potent antioxidant decreases risk of arthrosclerosis. Materials and Methods: This experimental study was conducted during 2015 in Iran. Cultured bovine aortic endothelial cells were incubated with hydrogen peroxide (H2O2) (20, 40 and 80 &micro;M) and Curcumin (10 &micro;M) for 24h. Then, the level of PDGF gene expression was analyzed by Real-Time PCR in untreated and treated cells. Results: The results demonstrated significant increase in the level of PDGF gene expression in H2O2 treated groups versus control. Also, treated groups with H2O2 -Curcumin showed notable decrease in the level of PDGF gene expression compared with H2O2 treated groups. &nbsp;Conclusion: Our results support valuable data about the application of Curcumin for protection against atherosclerosis

Inhibitory effect of Curcumin on phosphorylation NFκB-p65 induced by hydrogen peroxide in Bovine Endothelial Cells

Background & Objective: NF&kappa;B is a dimeric transcription factor with multiple subunits. Phosphorylation of&nbsp; p65 (one of NF&kappa;B subunits) by oxidative stress leads to the activation of NF&kappa;B. Imbalance between oxidative stress and cellular antioxidant capacity is the pathogenesis of many diseases. Consuming antioxidants in daily meal can be considered as a preventive strategy for inflammatory diseases. Among antioxidant components, curcumin is a natural polyphenol which is extracted from Tumeric. Curcumin can protect the human body from oxidative stress by neutralizing the free radicals. Material & Methods: Bovine Endothelial Cells (BECs) were treated with different concentration of H2O2 (10, 20, 80, 200 &mu;M). To investigate the inhibitory effect of curcumin on H2O2 mediated phosphorylation of p65, BECs were incubated with 5 and 10&mu;M concentration of curcumin. Protein concentration was measured by Bradford method and phosphorylation of p65 was assessed by western blotting. Results: Our finding indicated that p65 phosphorylation was increased two fold in presence of H2O2 (200 &mu;M) in comparison with control. The enhancing effect of H2O2 on p65 phosphorylation decreased at 30 min (P: 0.03) and 2 hours (P:0.015) after treatment with 10 &mu;M dose of curcumin. Conclusion: The result of this study indicates that one of anti-inflammatory mechanisms of curcumin is through NF&kappa;B pathway by inhibition of p65 subunit phosphorylation. &nbsp; &nbsp

The expression of lncRNAs CASC2, NEAT1, LINC00299 in breast cancer tissues and their relationship with the XBP1 splicing rate in Iranian patients during 2014–2019: A cross‐sectional study

Abstract Background and Aims Breast cancer is a leading cause of incidence and mortality in women globally. Identifying new molecular markers can aid in cancer diagnosis, targeted therapy, and treatment monitoring. This study aimed to measure the expression of the X‐box binding protein 1 (XBP1) gene, an index of the unfolded protein response (UPR), and long noncoding RNAs (lncRNAs), including Nuclear Enriched Abundant Transcript 1 (NEAT1), Cancer Susceptibility Candidate 2 (CASC2), and Long Intergenic Nonprotein Coding RNA 299 (LINC00299), as possible regulators of the UPR pathway. Methods Total RNA was extracted from 40 samples of breast tumor tissues and their respective controls. The expression level of lncRNAs CASC2, NEAT1, and LINC00299 was quantified using reverse transcription‐polymerase chain reaction (RT‐PCR). The ratio of the spliced form of XBP1 to its unspliced form (XBP1u) was determined by PCR and electrophoresis. Results The results showed a 2.8‐fold increase in the ratio of XBP1s/u in breast cancer tissues compared to adjacent nonmalignant samples (p < 0.05). Additionally, the level of lncRNAs NEAT1, CASC2, and LINC00299 in breast tumor tissues increased significantly by twofold, 1.5‐fold, and 2.3‐fold, respectively, compared to adjacent nonmalignant samples (p < 0.05). Conclusions Based on the association between the expression of lncRNAs CASC2, LINC00299, and NEAT1 and the XBP1s/u ratio, these lncRNAs could be potential regulators of the UPR pathway. Also, CASC2 and NEAT1 genes could be suggested as suitable biomarkers to distinguish cancerous tissue from noncancerous breast tissue due to their significant increase in expression in cancerous samples compared to adjacent noncancerous

Transforming growth factor–β1 mediated CHST11 and CHSY1 mRNA expression is ROS dependent in vascular smooth muscle cells

Transforming growth factor (TGF)-β1 mediates glycosaminoglycan (GAG) chain hyperelongation on secreted proteoglycans and these modifications are associated with increased lipid binding in the vessel wall and the development of atherosclerosis. In vascular smooth muscle cells (VSMCs), TGF-β1 regulated GAG elongation via extracellular signal-regulated kinase (ERK) and p38 as well as Smad2 linker region phosphorylation. In this study, our aim was to identify the TGF-β1 mediated signalling pathway involving reactive oxygen species (ROS) and Smad2 linker region phosphorylation that regulate the mRNA expression of GAG synthesizing enzymes, chondroitin 4-O-sulfotransferase 1 (CHST11) and chondroitin sulfate synthase 1 (CHSY1) which are the rate limiting enzymes involved in GAG chain elongation. Signalling molecules were assessed by western blotting, quantitative real-time PCR was used for analysis of gene expression and intracellular ROS level was measured by a fluorescence based assay. TGF-β1 induced ROS production in VSMCs. Nicotinamide adenine dinucleotide phosphate oxidase (Nox) inhibitors, diphenyleneiodonium (DPI) and apocynin blocked TGF-β1 mediated Smad2 linker region phosphorylation. TGF-β1 treatment increased the mRNA levels of CHST11 and CHSY1. Pharmacological inhibition of Nox blocked TGF-β1 mediated mitogen activated protein kinases (MAPKs) phosphorylation and TGF-β1 stimulated CHST11 and CHSY1 mRNA expression. These findings demonstrated that TGF-β1 mediated expression of CHST11 and CHSY1 can occur via Nox-dependent pathways and Smad2 linker region phosphorylation

Metabolic and hormonal effects of melatonin and/or magnesium supplementation in women with polycystic ovary syndrome: a randomized, double-blind, placebo-controlled trial

Abstract Background Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders among women of reproductive age. This study was designed to investigate the effects of melatonin and/or magnesium supplementation on metabolic profile and levels of sex hormones in PCOS women. Methods In an 8-week randomized double-blind placebo-controlled trial, 84 subjects with PCOS aged 18–40 years were randomly assigned based on the random block procedure to take magnesium, melatonin, magnesium plus melatonin, and placebo. Fasting blood samples were obtained at the beginning and end of the study. Results After intervention, the mean Pittsburg Sleep Quality Index score decreased significantly in both co-supplementation and melatonin groups (P < 0.001). Magnesium supplementation in combination with melatonin resulted in a significant greater decrease in testosterone concentrations compared with the placebo (P < 0.05). Co-supplementation of magnesium-melatonin had significantly reduced serum insulin levels (geometric means difference: − 1.11 (mIU/mL) (percent change: − 15.99)), homeostasis model of assessment-insulin resistance (HOMA-IR) (− 0.28 (− 18.66)), serum cholesterol (mean difference: − 16.08 (mg/dl) [95% CI − 24.24, − 7.92]), low-density lipoprotein cholesterol (LDL-C) − 18.96 (mg/dl) [− 28.73, − 9.20]) and testosterone levels (− 0.09 (ng/ml) (− 25.00)), as compared to the baseline values (P < 0.05). An increase in serum high-density lipoprotein cholesterol (HDL-C) levels was also observed following the administration of the melatonin alone (2.76 (mg/dl) [0.57, 4.95]) or in combination with magnesium (2.19 (mg/dl) [0.61, 3.77]) (P < 0.05). Conclusions Co-supplementation with magnesium and melatonin had beneficial effects on sleep quality and total testosterone. Additionally, melatonin supplementation alone was found to be associated with a significant reduction in PSQI score. Moreover, combined melatonin and magnesium supplementation was more effective in improving serum levels of cholesterol, LDL-C, HDL-C and insulin, and HOMA-IR. Trial registration: Iranian Registry of Clinical Trial. http://www.irct.ir : IRCT20191130045556N1, January 2020

Endothelin-1 (ET-1) stimulates carboxy terminal Smad2 phosphorylation in vascular endothelial cells by a mechanism dependent on ET receptors and de novo protein synthesis

Objective: G protein-coupled receptor (GPCR) agonists through their receptors can transactivate protein tyrosine kinase receptors such as epidermal growth factor receptor and serine/threonine kinase receptors most notably transforming growth factor (TGF)-β receptor (TβRI). This signalling mechanism represents a major expansion in the cellular outcomes attributable to GPCR signalling. This study addressed the role and mechanisms involved in GPCR agonist, endothelin-1 (ET-1)-mediated transactivation of the TβRI in bovine aortic endothelial cells (BAECs). Method: The in-vitro model used BAECs. Signalling intermediate phospho-Smad2 in the carboxy terminal was detected and quantified by Western blotting. Key finding: ET-1 treatment of BAECs resulted in a time and concentration-dependent increase in pSmad2C. Peak phosphorylation was evident with 100 nm treatment of ET-1 at 4–6 h. TβRI antagonist, SB431542 inhibited ET-1-mediated pSmad2C. In the presence of bosentan, a mixed ET and ET receptor antagonist ET-1-mediated pSmad2C levels were inhibited. The ET-mediated pSmad2C was blocked by the protein synthesis inhibitor, cycloheximide. Conclusion: In BAECs, ET-1 via the ETB receptor is involved in transactivation of the TβRI. The transactivation-dependent response is dependent upon de novo protein synthesis