Monica Raicu

Transcriptional regulation of NADPH oxidase isoforms, Nox1 and Nox4, by nuclear factor-κB in human aortic smooth muscle cells

Inflammation-induced changes in the activity and expression of NADPH oxidases (Nox) play a key role in atherogenesis. The molecular mechanisms of Nox regulation are scantily elucidated. Since nuclear factor-kappaB (NF-kappaB) controls the expression of many genes associated to inflammation-related diseases, in this study we have investigated the role of NF-kappaB signaling in the regulation of Nox1 and Nox4 transcription in human aortic smooth muscle cells (SMCs). Cultured cells were exposed to tumor necrosis factor-alpha (TNFalpha), a potent inducer of both Nox and NF-kappaB, up to 24h. Lucigenin-enhanced chemiluminescence and dichlorofluorescein assays, real-time polymerase chain reaction, and Western blot analysis showed that inhibition of NF-kappaB pathway reduced significantly the TNFalpha-dependent up-regulation of Nox-derived reactive oxygen species production, Nox1 and Nox4 expression. In silico analysis indicated the existence of typical NF-kappaB elements in the promoters of Nox1 and Nox4. Transient overexpression of p65/NF-kappaB significantly increased the promoter activities of both isoforms. Physical interaction of p65/NF-kappaB proteins with the predicted sites was demonstrated by chromatin immunoprecipitation assay. These findings demonstrate that NF-kappaB is an essential regulator of Nox1- and Nox4-containing NADPH oxidase in SMCs. Elucidation of the complex relationships between NF-kappaB and Nox enzymes may lead to a novel pharmacological strategy to reduce both inflammation and oxidative stress in atherosclerosis and its associated complications.

JAK/STAT Signaling Pathway Regulates Nox1 and Nox4-Based NADPH Oxidase in Human Aortic Smooth Muscle Cells

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 γ (IFNγ) 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 IFNγ-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. Conclusions—JAK/STAT is a key regulator of Nox1 and Nox4 in human vascular SMCs. Inhibition of JAK/STAT pathway and the consequent Nox-dependent oxidative stress may be an efficient therapeutic strategy to reduce atherogenesis.

AP-1–Dependent Transcriptional Regulation of NADPH Oxidase in Human Aortic Smooth Muscle Cells Role of p22phox Subunit

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)-&agr;. 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-&agr;-dependent upregulation of NADPHox activity and mRNA expression (NOX1, NOX4, p67phox, p47phox, p22phox). Inhibitors of AP-1 significantly diminished the Ang II or TNF-&agr;-stimulated p22phox promoter activity and protein level. Transient overexpression of c-Jun/c-Fos upregulated p22phox promoter activity. Transcription factor pull-down assay and chromatin immunoprecipitation demonstrated the physical interaction of c-Jun protein with predicted AP-1–binding sites in the p22phox gene promoter. Conclusions—In SMCs exposed to Ang II or TNF-&agr;, inhibition of AP-1–related pathways reduces NADPHox expression and the O2− production. The physical interaction of AP-1 with p22phox gene promoter facilitates NADPHox regulation.

Changes in oxidative balance in rat pericytes exposed to diabetic conditions

Recent data indicate that the oxidative stress plays an important role in the pathogenesis of diabetes and its complications such as retinopathy, nephropathy and accelerated atherosclerosis. In diabetic retinopathy, it was demonstrated a selective loss of pericytes accompanied by capillary basement membrane thickening, increased permeability and neovascularization. This study was designed to investigate the role of diabetic conditions such as high glucose, AGE‐Lysine, and angiotensin II in the modulation of antioxidant enzymes activities, glutathione level and reactive oxygen species (ROS) production in pericytes. The activity of antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total glutathione (GSH) was measured spectrophotometrically. The production of ROS was detected by spectrofluorimetry and fluorescence microscopy after loading the cells with 2′‐7′ dichlorofluoresceine diacetate; as positive control H2O2 was used. Intracellular calcium was determined using Fura 2 AM assay. The results showed that the cells cultured in high glucose alone, do not exhibit major changes in the antioxidant enzyme activities. The presence of AGE‐Lys or Ang II induced the increase of SOD activity. Their combination decreased significantly GPx activity and GSH level. Athree times increase in ROS production and a significant impairment of intracellular calcium homeostasis was detected in cells cultured in the presence of the three pro‐diabetic agents used. In conclusion, our data indicate that diabetic conditions induce in pericytes: (i) an increase of ROS and SOD activity, (ii) a decrease in GPx activity and GSH level, (iii) a major perturbation of the intracellular calcium homeostasis. The data may explain the structural and functional abnormalities of pericytes characteristic for diabetic retinopathy.

Effect of irreversibly glycated LDL in human vascular smooth muscle cells: lipid loading, oxidative and inflammatory stress

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.

Expression of functionally phagocyte-type NAD(P)H oxidase in pericytes: effect of angiotensin II and high glucose.

Background information. A growing body of evidence demonstrates the involvement of the oxidative stress in the development of vascular complications associated with diabetes, such as hypertension, retinopathy, nephropathy, neuropathy and atherosclerosis. However, the molecular mechanisms accountable for the increased production of reactive oxygen species (ROS) remain uncertain. Among others, the NAD(P)H oxidase is one of the most important sources of superoxide anion (O2−) that induce dysfunction of vascular cells. Pericytes (PCs) have an essential role in the capillary dysfunction in retinopathy and other vascular complications in diabetes. We questioned whether PCs express a functional phagocyte‐type NAD(P)H oxidase, and examined the role of angiotensin II and high glucose on the activity of the oxidase complex and expression of the essential subunit p22phox.

Tyrphostin AG490 reduces NAPDH oxidase activity and expression in the aorta of hypercholesterolemic apolipoprotein E-deficient mice

Oxidative stress-induced vascular injury represents a major contributor to the pathoetiology of atherosclerosis. Elevated NADPH oxidase (Nox) activity promotes oxidative injury of the cardiovascular cells. Janus-tyrosine-kinase (Jak) family regulate various aspects of the atherosclerotic process e.g., inflammation, cellular growth, proliferation, and migration. Here, we investigated the potential of Jak2 inhibition to counteract Nox-dependent O(2)(•-) formation in atherogenesis in hypercholesterolemic apolipoprotein E-deficient (ApoE(-/-)) mice. Male ApoE(-/-) mice fed a high-fat, cholesterol-rich diet were treated for 5 weeks with either vehicle or tyrphostin AG490 (1 mg/kg), a specific Jak2 inhibitor. Lucigenin-enhanced-chemiluminescence assay, real-time PCR and Western blot analysis revealed that Nox-derived O(2)(•-) generation, Nox1, Nox2, and Nox4 mRNA and protein levels were significantly elevated in the aortas of ApoE(-/-) mice fed a high-fat diet compared to ApoE(-/-) mice fed a normal diet. Treatment with tyrphostin AG490 significantly reduced the up-regulated Nox activity, the expression of each Nox subtype, as well as the protein level of CD68, a macrophage-specific marker. Morphometric analysis showed a marked reduction of atherosclerotic lesions in the aorta of AG490-treated animals. These data provide new insights into the regulation of vascular Nox by tyrphostins in the cardiovascular system. Since Jak2 transduces the signals of various cardiovascular risk factors, pharmacological manipulation of this signaling pathway may represent a novel strategy to reduce oxidative stress in atherosclerosis.

C/EBP transcription factors regulate NADPH oxidase in human aortic smooth muscle cells

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.

Advanced Glycation End Products, Oxidative Stress and Metalloproteinases are altered in the Cerebral Microvasculature during Aging

Biological aging is associated with an increased incidence of cerebrovascular disease. Recent findings indicate that oxidative stress promoting age-related changes of cerebral circulation are involved in neurodegenerative disorders such as Alzheimers disease (AD) and Parkinsons disease. The aim of this study was to evaluate the contribution of cerebral microvessels to the oxidative stress during brain aging, by: (i) assessment of precursors for advanced glycation end products (AGE) formation, (ii) activities of antioxidant enzymes, namely superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione disulfide reductase (GR), and (iii) the activities of metalloproteinases (MMPs), MMP-2 and MMP-9, involved in synaptogenesis and memory consolidation. The experiments were performed on two groups of male Wistar rats: 15 young (3-6 months old) and 15 aged (18-24 months old) animals. The cerebral microvessels were isolated by mechanical homogenization, the concentration of protein carbonyls and the activity of antioxidant enzymes were evaluated by spectrophotometry, and gelatin SDS-PAGE zymography was employed to evaluate MMP-2 and MMP-9 activities. The results showed that, by comparison with young rats, aged brain microvessels contain: (i) approximately 106 % increase of protein carbonyls production; (ii) approximately 68% higher GPx activity, unmodified activities of SOD and GR; (iii) approximately 30% diminishment in MMP-2 activity, and the specific occurrence of MMP-9 enzyme. The data suggest that the age-related changes of microvessels could increase the propensity for cerebral diseases and might represent, at least in part, a prerequisite for the deterioration of mental and physical status in the elderly.

Deleterious effects of nifedipine on smooth muscle cells implies alterations of intracellular calcium signaling

Nifedipine (NIF), a calcium channel blocker (CCB) from the first generation of dihydropyridines, induces detrimental effects on patients with cardiovascular diseases. We designed experiments to study, at cellular and molecular level, the mechanisms involved in the induction of deleterious effects by this drug. To this purpose, cultured human smooth muscle cells (HSMC) were used. The effect of NIF and two other CCB (FEL, AML) and inhibitors of intracellular signaling pathways (RR, TG, CAF and GEN) on intracellular calcium [Ca2+]I was determined by spectrofluorimetry using Fura 2 AM assay. The results showed that: (i) 10 μM NIF induced the increase of [Ca2+]I above the basal values (202.77 ± 23.98 nM vs. 48.68 ± 6.45 nM), an effect that was prevented by RR (50.45 ± 13.9 nM) and was not induced by the two other CCB; (ii) NIF had a thapsigargin‐like effect, because it induced the same release of intracellular calcium as TG (212.1 ± 25.62 nM); (iii) The response to NIF was reduced by 40% after the inhibition of IP3 receptor (121.21 ± 26.01 nM) and by 50% after the inhibition of tyrosine kinase (101.91 ± 7.76 nM). Together, these data demonstrate that NIF produces a deregulation of intracellular calcium homeostasis. The abnormal increase of [Ca2+]I is due to the activation of store operated channels from the plasma membrane responsible for capacitative calcium entry, a process modulated by the activity of tyrosine kinase and the Ca2+‐ATPase pump from the sarcoplasmic reticulum.