High glucose-induced increased expression of endothelin-1 in human endothelial cells is mediated by activated CCAAT/enhancer-binding proteins.

High glucose-induced endothelial dysfunction is partially mediated by the down-stream pathophysiological effects triggered by increased expression of endothelin-1 (ET-1). The molecular control mechanisms of ET-1 synthesis are yet to be discovered. Members of the CCAAT/enhancer-binding proteins (C/EBP) family are important regulators of key metabolic processes, cellular differentiation and proinflammatory genes. In this study, we aimed at elucidating the role of C/EBP in mediating the high glucose effect on ET-1 expression in human endothelial cells (EC). Human umbilical vein cells (EAhy926) and primary cultures of human aortic EC were exposed to high levels of glucose (16.5-25 mM). Real-time PCR, Western blot, enzyme-linked immunosorbent assay, ET-1 promoter-luciferase reporter analysis, and chromatin immunoprecipitation assays were employed to investigate ET-1 regulation. High glucose activated C/EBPα, C/EBPβ, and C/EBPδ in a dose-dependent manner. It also promoted significant increases in ET-1 gene and peptide expression. Chemical inhibition of JNK, p38MAPK and ERK1/2 diminished significantly the high glucose-induced nuclear translocation of C/EBP and ET-1 expression. Silencing of C/EBPα, C/EBPβ or C/EBPδ greatly reduced the high glucose-induced upregulation of ET-1 mRNA, pre-pro-ET-1, and ET-1 secretion. The expression of various C/EBP isoforms was selectively downregulated by siRNA-mediated gene silencing. In silico analysis indicated the existence of typical C/EBP elements within human ET-1 gene promoter. Transient overexpression of C/EBPα, C/EBPβ or C/EBPδ upregulated the luciferase level controlled by the ET-1 gene promoter. The direct interaction of C/EBPα, C/EBPβ or C/EBPδ proteins with the ET-1 promoter in high glucose-exposed EC was confirmed by chromatin immunoprecipitation assay. High glucose-induced ET-1 expression is mediated through multiple mechanisms. We present evidence that members of the C/EBP proinflammatory transcription factors are important regulators of ET-1 in high glucose-exposed human endothelial cells. High glucose-induced activation of C/EBP-related signaling pathways may induce excessive ET-1 synthesis, thus promoting vasoconstriction and dysfunction of the vascular wall cells in diabetes

C/EBPs mediate high glucose-induced (A) mRNA expression, (B) pre-pro-ET-1 level, and (C) ET-1 peptide secretion.

<p>(D) Representative immunoblots showing the pre-pro-ET-1 protein regulation. Quiescent EC were exposed (24 h) to 5.5 mM or 25 mM glucose in the absence/presence of siRNA sequences directed to silence the expression of various C/EBP subtypes. The ET-1 mRNA and protein expression were assayed by real time PCR, Western blot, and ELISA. n = 4, *<i>P</i><0.05, **<i>P</i><0.01. <i>P</i>-values were taken in relation to the C siRNA-transfected cells.</p

Evaluation of siRNA transfection and silencing efficiency in endothelial cells.

<p>Cultured cells were transfected with either control/scrambled (C siRNA), C/EBPα-, C/EBPβ- or C/EBPδ- siRNA. 48-hours after transfection (A) the up-take efficiency of FITC-siRNA was monitored by fluorescence microscopy (x20) and (B, C) the down-regulation of C/EBP protein levels by Western blot. n = 3.</p

Regulation of Nox enzymes expression in vascular pathophysiology: Focusing on transcription factors and epigenetic mechanisms

NADPH oxidases (Nox) represent a family of hetero-oligomeric enzymes whose exclusive biological function is the generation of reactive oxygen species (ROS). Nox-derived ROS are essential modulators of signal transduction pathways that control key physiological activities such as cell growth, proliferation, migration, differentiation, and apoptosis, immune responses, and biochemical pathways. Enhanced formation of Nox-derived ROS, which is generally associated with the up-regulation of different Nox subtypes, has been established in various pathologies, namely cardiovascular diseases, diabetes, obesity, cancer, and neurodegeneration. The detrimental effects of Nox-derived ROS are related to alterations in cell signalling and/or direct irreversible oxidative damage of nucleic acids, proteins, carbohydrates, and lipids. Thus, understanding of transcriptional regulation mechanisms of Nox enzymes have been extensively investigated in an attempt to find ways to counteract the excessive formation of Nox-derived ROS in various pathological states. Despite the numerous existing data, the molecular pathways responsible for Nox up-regulation are not completely understood. This review article summarizes some of the recent advances and concepts related to the regulation of Nox expression in the vascular pathophysiology. It highlights the role of transcription factors and epigenetic mechanisms in this process. Identification of the signalling molecules involved in Nox up-regulation, which is associated with the onset and development of cardiovascular dysfunction may contribute to the development of novel strategies for the treatment of cardiovascular diseases. Keywords: NADPH oxidase, Transcription factors, Epigenetics, Cardiovascular disease

High glucose-induced C/EBP activation is mediated by MAPK.

<p>Activation of (A) C/EBPα, (B) C/EBPβ, and (C) C/EBPδ was investigated by means of nuclear translocation as indicated under “Materials and Methods”. Quiescent EC were exposed (24 h) to either 5.5 mM or 25 mM glucose in the absence/presence of vehicle (DMSO) or MAPK pharmacological inhibitors (10 µM SP600125, 10 µM SB203580, 1 µM U0126). n = 3, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. <i>P</i>-values were taken in relation to the 25 mM glucose-exposed cells.</p

High glucose induces ET-1 gene transcription and activates C/EBPs.

<p>(A) Cells were transfected with either pET1-luc or pC/EBP-luc control plasmids as described under “Materials and Methods” in 5.5 mM-containing medium. Twenty-four hours after transfection cells were exposed for an additional 24 h to either 5.5 mM or 25 mM glucose. (B) Representative immunoblots depicting nuclear translocation of C/EBPα, C/EBPβ, and C/EBPδ in response to increasing concentrations of glucose (5.5 to 25 mM). (C) Densitometry analysis of C/EBP transcription factors activation. n = 3, *<i>P</i><0.05. <i>P</i>-values were taken in relation to the 5.5 mM glucose-exposed cells.</p

Inhibition of MAPK signaling blocks the high glucose-induced up-regulation of ET-1 transcription and mRNA expression.

<p>(A) Cultured cells were transfected with pET1-luc construct in 5.5 mM-containing medium. Twenty-four hours after transfection cells were exposed for an additional 24 h to either 5.5 mM or 25 mM glucose in the absence/presence of MAPK inhibitors (10 µM SP600125, 10 µM SB203580, 1 µM U0126). (B) Quiescent EC were exposed (24 h) to 5.5- or 25 mM glucose in the absence/presence of the indicated MAPK inhibitors and the mRNA was quantified by real-time PCR. n = 3, **<i>P</i><0.01, ***<i>P</i><0.001. <i>P</i>-values were taken in relation to the 25 mM glucose-exposed cells.</p

Functional analyses of C/EBP <i>cis</i>-acting elements within human ET-1 gene promoter.

<p>(A) Schematic depiction of the ET-1 gene promoter and the relative positions of the putative C/EBP binding sites are shown. (B) Analysis of ET-1 gene promoter activity in EC overexpressing C/EBPα, C/EBPβ or C/EBPδ. (C) Validation of C/EBPα, C/EBPβ or C/EBPδ overexpression by using pC/EBP-luc control plasmid. (D) ChIP assessment of C/EBPα-, C/EBPβ- or C/EBPδ – ET-1 promoter interaction in high glucose (25 mM)-exposed EC. IgG was used as negative control to test the specificity of the C/EBP antibodies. Representative agarose gel electrophoresis depicting the predicted amplicon size of the PCR products. (E) ChIP positive control - DNA fragment derived from human p21 gene promoter containing highly conserved C/EBP elements; ChIP negative control - DNA fragment derived from human c-Myc gene promoter that do not contain C/EBP sites. n = 3.</p

Epigenetic regulation of vascular NADPH oxidase expression and reactive oxygen species production by histone deacetylase-dependent mechanisms in experimental diabetes

Reactive oxygen species (ROS) generated by up-regulated NADPH oxidase (Nox) contribute to structural-functional alterations of the vascular wall in diabetes. Epigenetic mechanisms, such as histone acetylation, emerged as important regulators of gene expression in cardiovascular disorders. Since their role in diabetes is still elusive we hypothesized that histone deacetylase (HDAC)-dependent mechanisms could mediate vascular Nox overexpression in diabetic conditions. Non-diabetic and streptozotocin-induced diabetic C57BL/6J mice were randomized to receive vehicle or suberoylanilide hydroxamic acid (SAHA), a pan-HDAC inhibitor. In vitro studies were performed on a human aortic smooth muscle cell (SMC) line. Aortic SMCs typically express Nox1, Nox4, and Nox5 subtypes. HDAC1 and HDAC2 proteins along with Nox1, Nox2, and Nox4 levels were found significantly elevated in the aortas of diabetic mice compared to non-diabetic animals. Treatment of diabetic mice with SAHA mitigated the aortic expression of Nox1, Nox2, and Nox4 subtypes and NADPH-stimulated ROS production. High concentrations of glucose increased HDAC1 and HDAC2 protein levels in cultured SMCs. SAHA significantly reduced the high glucose-induced Nox1/4/5 expression, ROS production, and the formation malondialdehyde-protein adducts in SMCs. Overexpression of HDAC2 up-regulated the Nox1/4/5 gene promoter activities in SMCs. Physical interactions of HDAC1/2 and p300 proteins with Nox1/4/5 promoters were detected at the sites of active transcription. High glucose induced histone H3K27 acetylation enrichment at the promoters of Nox1/4/5 genes in SMCs. The novel data of this study indicate that HDACs mediate vascular Nox up-regulation in diabetes. HDAC inhibition reduces vascular ROS production in experimental diabetes, possibly by a mechanism involving negative regulation of Nox expression. Keywords: NADPH oxidase, Epigenetics, HDAC, Histone acetylation, Diabete