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Objective: In addition to the carboxy region, Smad2 transcription factor can be phosphorylated in the linker region as well. Phosphorylation of Smad2 linker region (Smad2L) promotes the expression of plasminogen activator inhibitor type 1 (PAI-1) which leads to cardiovascular disorders such as atherosclerosis. The purpose of this study was to evaluate the role of dual transactivation of EGF and TGF-β receptors in phosphorylation of Smad2L and protein expression of PAI-1 induced by endothelin-1 (ET-1) in bovine aortic endothelial cells (BAECs). In addition, as an intermediary of G protein-coupled receptor (GPCR) signaling, the functions of ROCK and PLC were investigated in dual transactivation pathways. Materials and Methods: The experimental study is an in vitro study performed on BAECs. Proteins were investigated by western blotting using protein-specific antibodies against phospho-Smad2 linker region residues (Ser245/250/255), phospho-Smad2 carboxy residues (465/467), ERK1/(Thr202/Thr204), and PAI-1. Results: TGF (2 ng/ml), EGF (100 ng/ml) and ET-1 (100 nM) induced the phosphorylation of Smad2L. This response was blocked in the presence of AG1478 (EGFR antagonists), SB431542 (TGFR inhibitor), and Y27632 (Rho-associated protein kinase (ROCK antagonist). Moreover, ET-1-increased protein expression of PAI-1 was decreased in the presence of bosentan (ET receptor inhibitor), AG1478, SB431542, and Y27632. Conclusion: The results indicated that ET-1 increases the phosphorylation of Smad2L and protein expression of PAI-1 via induced the transactivation pathways of EGFR and TGFR. This study is the first attempt to scrutinize the significant role of ROCK in the protein expression of PAI-1. [ABSTRACT FROM AUTHOR]

Background: Leptin and leptin receptor (Ob-R) are associated with worse prognosis, distant metastasis, and poor survival of breast cancer. We investigated the cytotoxic effect of silibinin and curcumin, individually and combined, on Ob-R expression in MCF-7 cells. Methods: This study was performed from October 2017 to April 2018 at the Department of Clinical Biochemistry, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. The cytotoxic effect of silibinin and curcumin, individually and combined, and their corresponding half- maximal inhibitory concentration (IC50) values were determined using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. The cells were treated with different concentrations of silibinin (50-400 µM), curcumin (10-35 µM), and their combinations for 24 and 48 hours. The expression of Ob-R was measured using the Western blot analysis by treating the cells with different concentrations of curcumin (10-25 µM), silibinin (50-250 µM), and their respective combinations. The difference in mean cell viability between the groups was calculated using one-way ANOVA followed by Tukey's post hoc test. Results: Silibinin and curcumin exerted time- and dose-dependent cytotoxic effect on MCF-7 cells. After treatment with silibinin, the IC50 values were about 250 and 50 µM at 24 and 48 hours, respectively. In terms of treatment with curcumin, the IC50 values were about 25 and 15 µM at 24 and 48 hours, respectively. Following treatment with silibinin, the Western blot analysis showed that Ob-R expression significantly decreased at 150 µM (P=0.031) and 200 µM (P=0.023) concentrations. Curcumin did not significantly decrease the Ob-R expression, however, the expression significantly decreased (P=0.004) when it was combined with silibinin. Conclusion: The combination of silibinin and curcumin significantly reduced Ob-R expression in MCF-7 cells compared with their individual effects. [ABSTRACT FROM AUTHOR]

BACKGROUND: Although the antioxidant properties of ginger have been revealed, there is little available information on the effectiveness of ginger on inflammatory disorders such as atherosclerosis. This study was carried out to examine the effect of ginger on improving the complication of atherosclerosis. METHODS: This study was a double-blind, placebo-controlled, randomized clinical trial conducted on patients with atherosclerosis. Participants in the ginger and control groups received 1600 mg of powdered ginger or placebo (wheat flour) in capsules daily for 8 weeks. Weight, body mass index (BMI), fasting blood sugar (FBS), cholesterol, triglyceride (TG), low-density lipoprotein (LDL), very-low-density lipoprotein (VLDL), high-density lipoprotein (HDL), lipoprotein (a) [Lp(a)], high-sensitivity C-reactive protein (hs-CRP), and total anti-oxidant capacity (TAC) were assessed before and after the intervention. RESULTS: Ginger consumption in the intervention group significantly reduced serum Lp(a) level (27.25 ± 1.30 ng/ml vs. 23.57 ± 0.97 ng/ml) (P = 0.040) and also the level of hs-CRP in the intervention group was 1.90 ± 0.33 μg/ml and 1.24 ± 0.15 μg/ml (P = 0.010) before and after intervention, respectively, but the levels of Lp(a) and hs-CRP were not decreased significantly in the placebo group. The level of TAC in the ginger group was 0.71 ± 0.05 mM and after the trial was 0.57 ± 0.04 mM (P = 0.050); no significant differences were seen in TAC when ginger was administered at 1600 mg/daily for 60 days. Also the level of Lp(a) and hs-CRP but not TAC reduced significantly in ginger group compared to placebo group after intervention. CONCLUSION: This study showed that ginger had anti-atherosclerosis and anti-glycemic properties associated through a significant decreased Lp(a) and FBS in patients with atherosclerosis supplemented with ginger. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (Tβ RI). 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 Tβ RI 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 n m treatment of ET-1 at 4-6 h. Tβ RI antagonist, SB431542 inhibited ET-1-mediated pSmad2C. In the presence of bosentan, a mixed ETA and ETB 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 Tβ RI. The transactivation-dependent response is dependent upon de novo protein synthesis. [ABSTRACT FROM AUTHOR]

Objective: According to the response-to-retention hypothesis, the inception of atherosclerosis is attributed to the deposition and retention of lipoprotein in the arterial intima, facilitated by altered proteoglycans with hyperelongated glycosaminoglycan (GAG) chains. Recent studies have elucidated a signaling pathway whereby transforming growth factor-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 known to exhibit pleiotropic effects, including antioxidant and anti-inflammatory. The purpose of the present research was to ascertain the influence of atorvastatin on TGF-β-stimulated expression of CHST11 and CHSY1 and associated signaling pathways using an in vitro model. Materials and Methods: In this experimental study, vascular smooth muscle cells (VSMCs) were pre-incubated with atorvastatin (0.1-10 µM) prior to being stimulated with TGF-β (2 ng/ml). The experiment aimed to evaluate the phosphorylation levels of Smad2C, Smad2L, ERK1/2, the NOX p47phox subunit, ROS production, and the mRNA expression of CHST11 and CHSY1. Results: Our research results indicated that atorvastatin inhibited TGF-β-stimulated CHSY1 and CHST11 mRNA expression. Further experiments showed that atorvastatin diminished TGF-β-stimulated ROS production and weakened TGF-β-stimulated phosphorylation of p47phox, ERK1/2, and Smad2L; however, we observed no effect on the TGF-βSmad2C pathway. Conclusion: These data suggest that atorvastatin demonstrates anti-atherogenic properties through the modulation of the ROS-ERK1/2-Smad2L signaling pathway. This provides valuable insight into the potential mechanisms by which atorvastatin exerts its pleiotropic effects against atherosclerosis. [ABSTRACT FROM AUTHOR]

Background: Transforming growth factor-β (TGF-β) in addition to the C-terminal region can phosphorylate receptorregulated 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. [ABSTRACT FROM AUTHOR]