Mechanisms underlying the radioprotective properties of γ-tocotrienol: comparative gene expression profiling in tocol-treated endothelial cells

Among the eight naturally occurring vitamin E analogs, γ-tocotrienol (GT3) is a particularly potent radioprophylactic agent in vivo. Moreover, GT3 protects endothelial cells from radiation injury not only by virtue of its antioxidant properties but also by inhibition of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase and by improving the availability of the nitric oxide synthase cofactor tetrahydrobiopterin. Nevertheless, the precise mechanisms underlying the superior radioprotective properties of GT3 compared with other tocols are not known. This study, therefore, examined the differences in gene expression profiles between GT3 and its tocopherol counterpart, γ-tocopherol, as well as between GT3 and α-tocopherol in human endothelial cells. Cells were treated with vehicle or the appropriate tocol for 24 h, after which total RNA was isolated and genome-wide gene expression profiles were obtained using the Illumina platform. GT3 was far more potent in inducing gene-expression changes than α-tocopherol or γ-tocopherol. In particular, GT3 induced multiple changes in pathways known to be of importance in the cellular response to radiation exposure. Affected GO functional clusters included response to oxidative stress, response to DNA damage stimuli, cell cycle phase, regulation of cell death, regulation of cell proliferation, hematopoiesis, and blood vessel development. These results form the basis for further studies to determine the exact importance of differentially affected GO functional clusters in endothelial radioprotection by GT3.

Electronic supplementary material

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CD40 signaling and Alzheimer's disease pathogenesis

The interaction between CD40 and its cognate ligand, CD40 ligand, is a primary regulator of the peripheral immune response, including modulation of T lymphocyte activation, B lymphocyte differentiation and antibody secretion, and innate immune cell activation, maturation, and survival. Recently, we and others have identified CD40 expression on a variety of CNS cells, including endothelial cells, smooth muscle cells, astroglia and microglia, and have found that, on many of these cells, CD40 expression is enhanced by pro-inflammatory stimuli. Importantly, the CD40–CD40 ligand interaction on microglia triggers a series of intracellular signaling events that are discussed, beginning with Src-family kinase activation and culminating in microglial activation as evidenced by tumor necrosis factor- secretion. Based on the involvement of microglial activation and brain inflammation in Alzheimer's disease pathogenesis, we have investigated co-stimulation of microglia, smooth muscle, and endothelial cells with CD40 ligand in the presence of low doses of freshly solubilized amyloid-β peptides. Data reviewed herein show that CD40 ligand and amyloid-β act synergistically to promote pro-inflammatory responses by these cells, including secretion of interleukin-1β by endothelial cells and tumor necrosis factor- by microglia. As these cytokines have been implicated in neuronal injury, a comprehensive model of pro-inflammatory CD40 ligand and amyloid-β initiated Alzheimer's disease pathogenesis (mediated by multiple CNS cells) is proposed.     

Regulatory network analyses reveal genome-wide potentiation of LIF signaling by glucocorticoids and define an innate cell defense response

While the hypothalamo-pituitary-adrenal axis (HPA) activates a general stress response by increasing glucocorticoid (Gc) synthesis, biological stress resulting from infections triggers the inflammatory response through production of cytokines. The pituitary gland integrates some of these signals by responding to the pro-inflammatory cytokines IL6 and LIF and to a negative Gc feedback loop. The present work used whole-genome approaches to define the LIF/STAT3 regulatory network and to delineate cross-talk between this pathway and Gc action. Genome-wide ChIP-chip identified 3,449 STAT3 binding sites, whereas 2,396 genes regulated by LIF and/or Gc were found by expression profiling. Surprisingly, LIF on its own changed expression of only 85 genes but the joint action of LIF and Gc potentiated the expression of more than a thousand genes. Accordingly, activation of both LIF and Gc pathways also potentiated STAT3 and GR recruitment to many STAT3 targets. Our analyses revealed an unexpected gene cluster that requires both stimuli for delayed activation; 83% of the genes in this cluster are involved in different cell defense mechanisms. Thus, stressors that trigger both general stress and inflammatory responses lead to activation of a stereotypic innate cellular defense response.     

Identification of human cell responses to benzene and benzene metabolites

Benzene is a common air pollutant and confirmed carcinogen, especially in reference to the hematopoietic system. In the present study we analyzed cytokine/chemokine production by, and gene expression induction in, human peripheral blood mononuclear cells upon their exposure to the benzene metabolites catechol, hydroquinone, 1,2,4-benzenetriol, and p-benzoquinone. Protein profiling showed that benzene metabolites can stimulate the production of chemokines, the proinflammatory cytokines TNF-alpha and IL-6, and the Th2 cytokines IL-4 and IL-5. Activated cells showed concurrent suppression of anti-inflammatory cytokine IL-10 expression. We also identified changes in global gene expression patterns in response to benzene metabolite challenges by using high-density oligonucleotide microarrays. Treatment with 1,2,4-benzenetriol resulted in the suppression of genes related to the regulation of protein expression and a concomitant activation of genes that encode heat shock proteins and cytochrome P450 family members. Protein and gene expression profiling identified unique human cellular responses upon exposure to benzene and benzene metabolites.     

Cytokines suppress the shear stress-stimulated release of vasoactive peptides from human endothelial cells

Endothelial cells from human umbilical vein perfused at 0.5 ml/min released vasopressin, endothelin, and substance P. Upon perfusion of the cells at 3.0 ml/min, the release of endothelia and vasopressin was significantly increased whereas the release of substance P was significantly decreased. Endothelial cells precultured for 24 h with interleukin-I (IL-1) and interferon-γ (IFN-γ) released more endothelin and less substance P at low flow and there was no further increase in release at high flow rate. These results suggest that cytokines suppress the normal responses of endothelial cells to increased fluid shear stress.     

Patterns of Living β-Actin Movement in Wounded Human Coronary Artery Endothelial Cells Exposed to Shear Stress

We previously demonstrated that physiologic levels of shear stress enhance endothelial repair. Cell spreading and migration, but not proliferation, were the major mechanisms accounting for the increases in wound closure rate (Albuquerque et al., 2000, Am. J. Physiol. Heart Circ. Physiol. 279, H293–H302). However, the patterns and movements of β-actin filaments responsible for cell motility and translocation in human coronary artery endothelial cells (HCAECs) have not been previously investigated under physiologic flow. HCAECs transfected with β-actin-GFP were cultured on type I collagen-coated coverslips. Confluent cell monolayers were subjected to laminar shear stress of 12 dynes/cm² for 18 h in a parallel-plate flow chamber to attain cellular alignment and then wounded by scraping with a metal spatula and subsequently exposed to a laminar shear stress of 20 dynes/cm² (S-W-sH) or static (S-W-sT) conditions. Time-lapse imaging and deconvolution microscopy was performed during the first 3 h after imposition of S-W-sH or S-W-sT conditions. The spatial and temporal dynamics of β-actin-GFP motility and translocation during wound closure in HCAEC monolayers were analyzed under both conditions. Compared with HCAEC under S-W-sT conditions, our data show that HCAEC under S-W-sH conditions demonstrated greater β-actin-GFP motility, filament and clumping patterns, and filament arcs used during cellular attachment and detachment. These findings demonstrate intriguing patterns of β-actin organization and movement during wound closure in HCAEC exposed to physiological flow.     

Regulation of gene expression by endogenous ABA in tomato plants

In response to water deficit, endogenous abscisic acid (ABA) accumulates in plants. This ABA serves as a signal for a multitude of processes, including regulation of gene expression. ABA accumulated in response to water deficit signals cellular as well as whole plant responses playing a role in the pattern of gene expression throughout the plant. Although the function of genes regulated by ABA during stress are currently poorly understood, a number of these genes may permit the plant to adapt to environmental stress.     

IL-8 and global gene expression analysis define a key role of ATP in renal epithelial cell responses induced by uropathogenic bacteria

The recent recognition of receptor-mediated ATP signalling as a pathway of epithelial pro-inflammatory cytokine release challenges the ubiquitous role of the TLR4 pathway during urinary tract infection. The aim of this study was to compare cellular responses of renal epithelial cells infected with uropathogenic Escherichia coli (UPEC) strain IA2 to stimulation with ATP-γ-S. A498 cells were infected or stimulated in the presence or absence of apyrase, that degrades extracellular ATP, or after siRNA-mediated knockdown of ATP-responding P2Y₂ receptors. Cellular IL-8 release and global gene expression were analysed. Both IA2 and A498 cells per se released ATP, which increased during infection. IA2 and ATP-γ-S caused a ∼5-fold increase in cellular release of IL-8 and stimulations performed in the presence of apyrase or after siRNA knockdown of P2Y₂ receptors resulted in attenuation of IA2-mediated IL-8 release. Microarray results show that both IA2 and ATP-γ-S induced marked changes in gene expression of renal cells. Thirty-six genes were in common between both stimuli, and many of these are key genes belonging to classical response pathways of bacterial infection. Functional analysis shows that 88 biological function-annotated cellular pathways were identical between IA2 and ATP-γ-S stimuli. Results show that UPEC-induced release of IL-8 is dependent on P2Y₂ signalling and that cellular responses elicited by UPEC and ATP-γ-S have many identical features. This indicates that renal epithelial responses elicited by bacteria could be mediated by bacteria- or host-derived ATP, thus defining a key role of ATP during infection.

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The online version of this article (doi:10.1007/s11302-014-9414-7) contains supplementary material, which is available to authorized users.