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.

Objective-NADPH oxidase (NADPHox) is the major source of reactive oxygen species in vascular diseases; the mechanisms of enzyme activation are not completely elucidated. AP-1 controls the expression of many genes linked to vascular smooth muscle cells (SMCs) dysfunction. In this study we searched for the role of AP-1 in the regulation of NADPHox expression and function in human aortic SMCs exposed to proinflammatory conditions. Methods and Results-Cultured SMCs were exposed to either angiotensin II (Ang II) or tumor necrosis factor (TNF)-␣.The lucigenin-enhanced chemiluminescence assay and real-time polymerase chain reaction analysis revealed that AP-1 and mitogen-activated protein kinase inhibitors reduced both Ang II or TNF-␣-dependent upregulation of NADPHox activity and mRNA expression (NOX1, NOX4, p67 phox , p47 phox , p22 phox ). n cardiovascular disorders such as hypertension, atherosclerosis, heart failure, and diabetes the generation of reactive oxygen species (ROS) is increased in the vasculature primarily through the activation of NADPH oxidase 1,2 (NADPHox), a group of multi-subunit enzymes expressed by endothelial cells, smooth muscle cells (SMCs), pericytes, adventitial fibroblasts, and cardiac myocytes. 3,4 Hypertension, a major risk factor for cardiovascular diseases, is associated with functional-structural changes of blood vessels and in particular with vascular SMC hypertrophy, synthesis of excess extracellular matrix, and inflammatory cytokines. 5,6 Evidence exists that angiotensin II (Ang II) plays an important role in the pathogenesis of hypertensionrelated cardiovascular diseases. Besides its vasoactive action, Ang II stimulates NADPHox-derived ROS production, and exerts hypertrophic and hyperplasic effects by activating various intracellular signal transduction pathways. The latter include mitogen-activated protein kinase (MAPK) family members, extracellular signal-regulated protein kinase (ERK)1/2, c-Jun amino terminal kinase (JNK), p38 MAPK, and transcription factors such as nuclear factor kB (NF-kB) and activator protein-1 (AP-1). [7][8][9][10] Also, NADPHox-resulting ROS activate AP-1, which regulates cell growth and transformation, inflammation, innate immune response, and apoptosis. In vivo, evidence supports a key role of AP-1 in the vascular response to injury. [7][8][9][10][11][12][13][14][15] The NADPHox complex, the major source of superoxide in the vascular wall, 16 consists of 5 subunits: a membraneassociated cytochrome b 558 containing gp91 phox and p22 phox and a cytosolic complex of p40 phox , p47 phox , p67 phox17 . Besides gp91 phox (NOX2), NOX1 and NOX4 were identified in cardiovascular cells 3,4 and all require p22 phox for their activity. 6,18,19 The increased expression of oxidase subunits correlates with an enhanced vascular superoxid production in human atherosclerotic arteries and in hypertension 18,20 ; although important, the transcriptional regulatory mechanisms of NADPHox components are not entirely elucidated.Because proinflammatory stimuli activate both AP-1...

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.

No abstract

NADPH oxidase (Nox)-derived reactive oxygen species (ROS) are instrumental in all inflammatory phases of atherosclerosis. Dysregulated histone deacetylase (HDAC)-related epigenetic pathways have been mechanistically linked to alterations in gene expression in experimental models of cardiovascular disorders. Hitherto, the relation between HDAC and Nox in atherosclerosis is not known. We aimed at uncovering whether HDAC plays a role in mediating Nox up-regulation, oxidative stress, inflammation, and atherosclerotic lesion progression. Human non-atherosclerotic and atherosclerotic arterial samples, ApoE−/− mice, and in vitro polarized monocyte-derived M1/M2-macrophages (Mac) were examined. Male ApoE−/− mice, maintained on normal or high-fat, cholesterol-rich diet, were randomized to receive 10 mg/kg suberoylanilide hydroxamic acid (SAHA), a pan-HDAC inhibitor, or its vehicle, for 4 weeks. In the human/animal studies, real-time PCR, Western blot, lipid staining, lucigenin-enhanced chemiluminescence assay, and enzyme-linked immunosorbent assay were employed. The protein levels of class I, class IIa, class IIb, and class IV HDAC isoenzymes were significantly elevated both in human atherosclerotic tissue samples and in atherosclerotic aorta of ApoE−/− mice. Treatment of ApoE−/− mice with SAHA reduced significantly the extent of atherosclerotic lesions, and the aortic expression of Nox subtypes, NADPH-stimulated ROS production, oxidative stress and pro-inflammatory markers. Significantly up-regulated HDAC and Nox subtypes were detected in inflammatory M1-Mac. In these cells, SAHA reduced the Nox1/2/4 transcript levels. Collectively, HDAC inhibition reduced atherosclerotic lesion progression in ApoE−/− mice, possibly by intertwined mechanisms involving negative regulation of Nox expression and inflammation. The data propose that HDAC-oriented pharmacological interventions could represent an effective therapeutic strategy in atherosclerosis.

No abstract

Emerging evidence demonstrates the involvement of endothelin-1 (ET-1) in the pathophysiology of cardiovascular disorders associated with diabetes mellitus. The molecular mechanisms accountable for the increased production of ET-1 are not completely defined. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway is an essential pathogenic mechanism leading to endothelial cell dysfunction. Our aim has been to investigate the role of JAK/STAT in the regulation of ET-1 synthesis in human endothelial cells (EAhy926 cells line). EAhy926 cells were exposed to normal (5 mM) or high (25 mM) glucose concentrations in the presence/absence of various JAK/STAT inhibitors. Using real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and gene reporter assay, we found that JAK/STAT inhibitors (STAT1 decoy oligodeoxynucleotides, AG490, S3I201, WP1066) significantly diminished the high-glucose-dependent up-regulation of ET-1 mRNA, peptide synthesis, and promoter activity. In silico analysis of the human ET-1 promoter revealed the presence of typical STAT1-gamma-activated sequence (STAT1-GAS) elements. Transient overexpression of STAT1 indicated an up-regulation of ET-1 promoter activity. Chromatin immunoprecipitation demonstrated the physical interaction of STAT1 proteins with the predicted GAS sites. Regulation of ET-1 synthesis by the JAK/STAT pathway thus represents a novel mechanism by which high glucose induces endothelial cell dysfunction in diabetes. Since the JAK/STAT system is an important regulator of the response of endothelial cells to injury, the modulation of this system and the subsequent decrease in ET-1 level may represent a key pharmacological target in diabetes-associated cardiovascular disorders.

In atherosclerosis, oxidative stress-induced vascular smooth muscle cells (SMCs) dysfunction is partially mediated by up-regulated NADPH oxidase (Nox); the mechanisms of enzyme regulation are not entirely defined. CCAAT/enhancer-binding proteins (C/EBP) regulate cellular proliferation and differentiation, and the expression of many inflammatory and immune genes. We aimed at elucidating the role of C/EBP in the regulation of Nox in SMCs exposed to pro-inflammatory conditions. Human aortic SMCs were treated with interferon-γ (IFN-γ) for up to 24 hrs. Lucigenin-enhanced chemiluminescence, real-time PCR, Western blot, promoter-luciferase reporter analysis and chromatin immunoprecipitation assays were employed to investigate Nox regulation. IFN-γ dose-dependently induced Nox activity and expression, nuclear translocation and up-regulation of C/EBPα, C/EBPβ and C/EBPδ protein expression levels. Silencing of C/EBPα, C/EBPβ or C/EBPδ reduced significantly but differentially the IFN-γ-induced up-regulation of Nox activity, gene and protein expression. In silico analysis indicated the existence of typical C/EBP sites within Nox1, Nox4 and Nox5 promoters. Transient overexpression of C/EBPα, C/EBPβ or C/EBPδ enhanced the luciferase level directed by the promoters of the Nox subtypes. Chromatin immunoprecipitation demonstrated the physical interaction of C/EBPα, C/EBPβ and C/EBPδ proteins with the Nox1/4/5 promoters. C/EBP transcription factors are important regulators of Nox enzymes in IFN-γ-exposed SMCs. Activation of C/EBP may induce excessive Nox-derived reactive oxygen species formation, further contributing to SMCs dysfunction and atherosclerotic plaque development. Pharmacological targeting of C/EBP-related signalling pathways may be used to counteract the adverse effects of oxidative stress.