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Objective-Oxidative stress mediated by Nox1-and Nox4-based NADPH oxidase (Nox) plays a key role in vascular diseases. The molecular mechanisms involved in the regulation of Nox are not entirely elucidated. Because JAK/STAT regulates many genes linked to inflammation, cell proliferation, and differentiation, we questioned whether this pathway is involved in the regulation of Nox1 and Nox4 in human aortic smooth muscle cells (SMCs). Methods and Results-Cultured SMCs were exposed to interferon ␥ (IFN␥) for 24 hours. Using lucigenin-enhanced chemiluminescence and dihydroethidium assays, real-time polymerase chain reaction, and Western blot analysis, we found that JAK/STAT inhibitors significantly diminished the IFN␥-dependent upregulation of Nox activity, Nox1 and Nox4 expression. In silico analysis revealed the presence of highly conserved GAS elements within human Nox1, Nox4, p22phox, p47phox, and p67phox promoters. Transient overexpression of STAT1/STAT3 augmented the promoter activities of each subunit. JAK/STAT blockade reduced the Nox subunits transcription. Chromatin immunoprecipitation demonstrated the physical interaction of STAT1/STAT3 proteins with the predicted GAS elements from Nox1 and Nox4 promoters. Key Words: NADPH oxidase Ⅲ JAK/STAT Ⅲ oxidative stress Ⅲ atherosclerosis E merging clinical and experimental evidence demonstrate the role of oxidative stress in the development of cardiovascular disorders. Reactive oxygen species (ROS) are implicated in different cell processes associated to vascular plaque formation such as growth, proliferation, differentiation, and apoptosis of smooth muscle cells (SMCs). 1,2 Deciphering the molecular mechanisms underlying the regulation of ROS production may lead to new therapeutic approaches. Conclusions-JAK/STATNADPH oxidases (Nox) are regulated by a plethora of stimuli and represent a major source of ROS in the vasculature. Depending on the cell type, the vascular Nox comprises 3 distinct catalytic subunits (Nox1, Nox2, Nox4) and 6 cytosolic regulatory components (p47phox, p67phox, NoxO1, NoxA1, Rac1/2). The p22phox component is essential for Nox activity forming a bound complex with 4 SMCs express predominantly Nox1 and Nox4 isoforms, which are differentially distributed in the cellular compartments and direct several redox-dependent processes. 5 It was postulated that Nox1 associates with and promotes SMCs proliferation, whereas Nox4 is required for the maintenance of differentiated phenotype. 6 Changes in gene expression of the Nox isoforms are critical for its function. In previous studies we showed that the expression of p22phox subunit and the ensuing superoxide production is regulated by NF-kB and AP-1 in human aortic SMCs. 7,8 However, the transcriptional regulatory mechanisms of oxidase components are not entirely elucidated.Activation of Janus tyrosine kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway is an essential pathogenic mechanism leading to SMC hypertrophy and hyperplasia. It was shown that STAT1 and STAT...

Reactive oxygen species (ROS) are essential mediators of normal cell physiology. However, in the last few decades, it has become evident that ROS overproduction and/or alterations of the antioxidant system associated with inflammation and metabolic dysfunction are key pathological triggers of cardiovascular disorders. NADPH oxidases (Nox) represent a class of hetero-oligomeric enzymes whose primary function is the generation of ROS. In the vasculature, Nox-derived ROS contribute to the maintenance of vascular tone and regulate important processes such as cell growth, proliferation, differentiation, apoptosis, cytoskeletal organization, and cell migration. Under pathological conditions, excessive Nox-dependent ROS formation, which is generally associated with the up-regulation of different Nox subtypes, induces dysregulation of the redox control systems and promotes oxidative injury of the cardiovascular cells. The molecular mechanism of Nox-derived ROS generation and the means by which this class of molecule contributes to vascular damage remain debatable issues. This review focuses on the processes of ROS formation, molecular targets, and neutralization in the vasculature and provides an overview of the novel concepts regarding Nox functions, expression, and regulation in vascular health and disease. Because Nox enzymes are the most important sources of ROS in the vasculature, therapeutic perspectives to counteract Nox-dependent oxidative stress in the cardiovascular system are discussed.

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...

The review pinpoints operational concepts related to the redox biology network applied to the pathophysiology and therapeutics of solid tumors. A sophisticated network of intrinsic and extrinsic cues, integrated in the tumor niche, drives tumorigenesis and tumor progression. Critical mutations and distorted redox signaling pathways orchestrate pathologic events inside cancer cells, resulting in resistance to stress and death signals, aberrant proliferation and efficient repair mechanisms. Additionally, the complex inter-cellular crosstalk within the tumor niche, mediated by cytokines, redox-sensitive danger signals (HMGB1) and exosomes, under the pressure of multiple stresses (oxidative, inflammatory, metabolic), greatly contributes to the malignant phenotype. The tumor-associated inflammatory stress and its suppressive action on the anti-tumor immune response are highlighted. We further emphasize that ROS may act either as supporter or enemy of cancer cells, depending on the context. Oxidative stress-based therapies, such as radiotherapy and photodynamic therapy, take advantage of the cytotoxic face of ROS for killing tumor cells by a non-physiologically sudden, localized and intense oxidative burst. The type of tumor cell death elicited by these therapies is discussed. Therapy outcome depends on the differential sensitivity to oxidative stress of particular tumor cells, such as cancer stem cells, and therefore co-therapies that transiently down-regulate their intrinsic antioxidant system hold great promise. We draw attention on the consequences of the damage signals delivered by oxidative stress-injured cells to neighboring and distant cells, and emphasize the benefits of therapeutically triggered immunologic cell death in metastatic cancer. An integrative approach should be applied when designing therapeutic strategies in cancer, taking into consideration the mutational, metabolic, inflammatory and oxidative status of tumor cells, cellular heterogeneity and the hypoxia map in the tumor niche, along with the adjoining and systemic effects of oxidative stress-based therapies.

Accumulating evidence demonstrates the involvement of oxidative stress in the pathophysiology of cardiovascular diseases. The molecular mechanisms accountable for the increased production of reactive oxygen species remain uncertain. Among others, NADPH oxidase is one of the most important sources of superoxide in vascular cells. Here we investigate the role of NF-kB in the regulation of p22(phox) subunit and NADPH oxidase activity, in human aortic smooth muscle cells. Overexpression of p65/RelA or IKKbeta up-regulated p22(phox) gene promoter activity. Transcription factor pull-down assays demonstrated the physical interaction of p65/RelA protein with predicted NF-kB binding sites. Real time PCR and Western blotting analysis showed that p22(phox) mRNA and protein expression are significantly down-regulated by NF-kB decoy oligodeoxynucleotides and N-alpha-tosyl-l-phenylalanine chloromethyl ketone (TPCK). Lucigenin-enhanced chemiluminescence assay revealed that NF-kB inhibitors reduce the NADPH-dependent superoxide production. Regulation of NADPH oxidase by NF-kB may represent a possible mechanism whereby pro-inflammatory factors induce oxidative stress in atherosclerosis, hypertension, diabetes, stroke or heart failure.

The major complication of diabetes is accelerated atherosclerosis, the progression of which entails complex interactions between the modified low-density lipoproteins (LDL) and the cells of the arterial wall. Advanced glycation end product-modified-LDL (AGE-LDL) that occurs at high rate in diabetes contributes to diabetic atherosclerosis, but the underlying mechanisms are not fully understood. The aim of this study was to assess the direct effect of AGE-LDL on human vascular smooth muscle cells (hSMC) dysfunction. Cultured hSMC incubated (24 hrs) with human AGE-LDL, native LDL (nLDL) or oxidized LDL (oxLDL) were subjected to: (i) quantification of the expression of the receptors for modified LDL and AGE proteins (LRP1, CD36, RAGE) and estimation of lipid loading, (ii) determination of NADPH oxidase activity and reactive oxygen species (ROS) production and (iii) evaluation of the expression of monocyte chemoattractant protein-1 (MCP-1). The results show that exposure of hSMC to AGE-LDL (compared to nLDL) induced: (a) increased NADPH oxidase activity (30%) and ROS production (28%) by up-regulation of NOX1, NOX4, p22phox and p67phox expression, (b) accumulation of intracellular cholesteryl esters, (c) enhanced gene expression of LRP1 (160%) and CD36 (35%), and protein expression of LRP1, CD36 and RAGE, (d) increased MCP-1 gene expression (160%) and protein secretion (300%) and (e) augmented cell proliferation (30%). In conclusion, AGE-LDL activates hSMC (increasing CD36, LRP1, RAGE), inducing a pro-oxidant state (activation of NADPHox), lipid accumulation and a pro-inflammatory state (expression of MCP-1). These results may partly explain the contribution of AGE-LDL and hSMC to the accelerated atherosclerosis in diabetes.