Gene Expression Profiling of Cultured Cells From Brainstem of Newborn Spontaneously Hypertensive and Wistar Kyoto Rats

The spontaneously hypertensive rat (SHR) is a good model to study several diseases such as the attention-deficit hyperactivity disorder, cardiopulmonary impairment, nephropathy, as well as hypertension, which is a multifactor disease that possibly involves alterations in gene expression in hypertensive relative to normotensive subjects. In this study, we used high-density oligoarrays to compare gene expression profiles in cultured neurons and glia from brainstem of newborn normotensive Wistar Kyoto (WKY) and SHR rats. We found 376 genes differentially expressed between SHR and WKY brainstem cells that preferentially map to 17 metabolic/signaling pathways. Some of the pathways and regulated genes identified herein are obviously related to cardiovascular regulation; in addition there are several genes differentially expressed in SHR not yet associated to hypertension, which may be attributed to other differences between SHR and WKY strains. This constitute a rich resource for the identification and characterization of novel genes associated to phenotypic differences observed in SHR relative to WKY, including hypertension. In conclusion, this study describes for the first time the gene profiling pattern of brainstem cells from SHR and WKY rats, which opens up new possibilities and strategies of investigation and possible therapeutics to hypertension, as well as for the understanding of the brain contribution to phenotypic differences between SHR and WKY rats.

Phenotypic modulation of cultured vascular smooth muscle cells: a functional analysis focusing on MLC and ERK1/2 phosphorylation

Primary cultures of vascular smooth muscle cells (VSMCs) from rats offer a good model system to examine the molecular basis of mechanism of vascular contraction-relaxation. However, during pathological conditions such as atherosclerosis and hypertension, VSMCs characteristically exhibit phenotypic modulation, change from a quiescent contractile to a proliferative synthetic phenotype, which impairs this mechanism of vascular contraction-relaxation. Taking in account that Myosin light chain (MLC) and ERK1/2 directly participate in the process of vascular contraction, the aim of the current study was to analyze the involvement of MLC and ERK1/2 signaling during the process of VSMCs phenotypic modulation. Primary cultures of VSMCs from rat thoracic aortas were isolated and submitted to different number of passages or to freezing condition. Semi-quantitative RT-PCR was used to evaluate the mRNA levels of VSMCs differentiation markers, and western blot assays were used to determine the MLC and ERK1/2 phosphorylation levels during VSMCs phenotypic modulation. Also, immunocytochemical experiments were performed to evaluate morphological alterations occurred during the phenotypic modulation. Elevated number of passages (up to 4) as well as the freezing/thawing process induced a significant phenotypic modulation in VSMCs, which was accompanied by diminished MLC and ERK1/2 phosphorylation levels. Phosphorylation of MLC was suppressed completely by the treatment with a synthetic inhibitor of MEK-1, a direct upstream of ERK1/2, PD98059. These findings provide that ERK1/2-promoted MLC phosphorylation is impaired during VSMCs phenotypic modulation, suggesting that ERK1/2 signaling pathway may represent a potential target for understanding the pathogenesis of several vascular disease processes frequently associated to this condition.

Amyloid beta-peptide activates nuclear factor-kappa B through an N-methyl-D-aspartate signaling pathway in cultured cerebellar cells

Amyloid P-peptide (A beta) likely causes functional alterations in neurons well prior to their death. Nuclear factor-kappa B (NF-kappa B), a transcription factor that is known to play important roles in cell survival and apoptosis, has been shown to be modulated by A beta in neurons and glia, but the mechanism is unknown. Because A beta has also been shown to enhance activation of N-methyl-D-aspartate (NMDA) receptors, we investigated the role of NMDA receptor-mediated intracellular signaling pathways in A beta-induced NF-kappa B activation in primary cultured rat cerebellar cells. Cells were treated with different concentrations of A beta 1-40 (1 or 2 mu M) for different periods (6, 12, or 24 hr). MK-801 (NMDA antagonist), manumycin A and FTase inhibitor 1 (farnesyltransferase inhibitors), PP1 (Src-family tyrosine kinase inhibitor), PD98059 [mitogen-activated protein kinase (MAPK) inhibitor], and LY294002 [phosphatidylinositol 3-kinase (PI3-k) inhibitor] were added 20 min before A beta treatment of the cells. A beta induced a time- and concentration-dependent activation of NF-kappa B (1 mu M, 12 hr); both p50/p65 and p50/p50 NF-kappa B dimers were involved. This activation was abolished by MK-801 and attenuated by manumycin A, FTase inhibitor 1, PP1, PD98059, and LY294002. AP at 1 mu M increased the expression of inhibitory protein I kappa B, brain-derived neurotrophic factor, inducible nitric oxide synthase, tumor necrosis factor-alpha, and interleukin-1 beta as shown by RTPCR assays. Collectively, these findings suggest that AP activates NF-kappa B by an NMDA-Src-Ras-like protein through MAPK and PI3-k pathways in cultured cerebellar cells. This pathway may mediate an adaptive, neuroprotective response to A beta. (c) 2007 Wiley-Liss, Inc.

Anticlastogenic effect of β-glucan, extracted from Saccharomyces cerevisiae, on cultured cells exposed to ultraviolet radiation

β-glucan is an important polysaccharide due to its medicinal properties of stimulating the immune system and preventing chronic diseases such as cancer. The aim of the present study was to determine the anticlastogenic effect of β-glucan in cells exposed to ultraviolet radiation (UV). Chromosome aberration assay was performed in drug-metabolizing cells (HTC) and non drug-metabolizing cells (CHO-K1 and repair-deficient CHO-xrs5), using different treatment protocols. Continuous treatment (UV + β-glucan) was not effective in reducing the DNA damage only in CHO-xrs5 cells. However, the pre-treatment protocol (β-glucan before UV exposition) was effective in reducing DNA damage only in CHO-K1 cells. In post-treatment (β-glucan after UV exposition) did not show significative anticlastogenic effects, although there was a tendency toward prevention. The data suggest that β-glucan has more than one action mechanism, being capable of exerting desmutagenic as well as bio-antimutagenic action. The findings also suggest that the presence of the xenobiotic metabolizing system can reduce the chemopreventive capacity of β-glucan. Therefore, these results indicate that β-glucan from Saccharomyces cerevisiae can be used in the prevention and/or reduction of DNA damage. © 2012 Springer Science+Business Media B.V.

Chemoprotective activity of the isoflavones, genistein and daidzein on mutagenicity induced by direct and indirect mutagens in cultured HTC cells

Isoflavones are phenolic compounds widely distributed in plants and found in a high percentage in soybeans. They have important biological properties and are regarded as potential chemopreventive agents. The aim of this study was to verify the preventive effect of two soy isoflavones (genistein and daidzein) by a micronucleus assay, analysis of GST activity, and real-time RT-PCR analysis of GSTa2 gene expression. Mutagens of direct (doxorubicin) and indirect (2-aminoanthracene) DNA damage were used. Hepatoma cells (HTC) were treated with genistein or daidzein for 26 h at noncytotoxic concentrations; 10 μM when alone, and 0.1, 1.0 and 10 μM when combined with genotoxic agents. The micronucleus test demonstrated that both isoflavones alone had no genotoxic effect. Genistein showed antimutagenic effects at 10 μM with both direct and indirect DNA damage agents. On phase II enzyme regulation, the current study indicated an increase in total cytoplasmic GST activity in response to genistein and daidzein at 10 μM supplementation. However, the mRNA levels of GSTa2 isozymes were not differentially modulated by genistein or daidzein. The results point to an in vitro antimutagenic activity of genistein against direct and indirect DNA damage-induced mutagenicity. © 2012 Springer Science+Business Media B.V.

Cytotoxicity of adhesive systems of different hydrophilicities on cultured odontoblast-like cells

This study evaluated the cytotoxicity of experimental adhesive systems (EASs) on odontoblast-like cells. Paper discs (n=132) were impregnated with 10 μL of each EAS-R1, R2, R3, R4, and R5 (in an ascending order of hydrophilicity), followed by photoactivation. R1 and R2 are nonsolvated hydrophobic blends, R3 represents a simplified etch-and-rinse adhesive system, and R4 and R5 represent simplified self-etch adhesive systems. Discs were immersed in Dulbecco's modified Eagle's medium for 24 h to obtain eluates applied on MDPC-23 cell cultures. No material was applied on discs used as control (R0). Cell viability [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay], total protein (TP) production, alkaline phosphatase (ALP) activity, type of cell death, and degree of monomer conversion Fourier transform infrared (%DC-FTIR) were evaluated. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests (α=0.05). Considering R0 (control) as having 100% of cell viability, R1, R2, R3, R4, and R5 reduced the metabolic activity of cells by 36.4, 3.1, 0.2, 21.5, and 65.7%, respectively, but only R1 and R5 differed from R0. Comparing with R0, lower TP production was observed for R1, R4, and R5, while ALP activity decreased for R1 and R5. Necrotic cell death was predominant for all EASs, but only R1, R4, and R5 differed from R0. Only R5 presented a different apoptotic cell death ratio from R0. R1 presented the lowest %DC (ca. 37%), whereas R4 and R5 presented the highest (ca. 56%). In conclusion, R2 and R3 were not toxic to the MDPC-23 cells, suggesting that the degree of hydrophilicity or %DC of the EASs alone were not responsible for their cytopathic effects. © 2013 Wiley Periodicals, Inc.