Pharmacological inhibition of histone deacetylase reduces NADPH oxidase expression, oxidative stress and the progression of atherosclerotic lesions in hypercholesterolemic apolipoprotein E-deficient mice; potential implications for human atherosclerosis.

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.

Histone Acetyltransferase-Dependent Pathways Mediate Upregulation of NADPH Oxidase 5 in Human Macrophages under Inflammatory Conditions: A Potential Mechanism of Reactive Oxygen Species Overproduction in Atherosclerosis.

Histone acetylation plays a major role in epigenetic regulation of gene expression. Monocyte-derived macrophages express functional NADPH oxidase 5 (Nox5) that contributes to oxidative stress in atherogenesis. The mechanisms of Nox5 regulation are not entirely elucidated. The aim of this study was to investigate the expression pattern of key histone acetyltransferase subtypes (p300, HAT1) in human atherosclerosis and to determine their role in mediating the upregulation of Nox5 in macrophages under inflammatory conditions. Human nonatherosclerotic and atherosclerotic tissue samples were collected in order to determine the expression of p300 and HAT1 isoforms, H3K27ac, and Nox5. In vitro determinations were done on human macrophages exposed to lipopolysaccharide in the absence or presence of histone acetyltransferase inhibitors. Western blot, immunohistochemistry, immunofluorescence, real-time PCR, transfection, and chromatin immunoprecipitation assay were employed. The protein levels of p300 and HAT1 isoforms, H3K27ac, and Nox5 were found significantly elevated in human atherosclerotic specimens. Immunohistochemistry/immunofluorescence staining revealed that p300, HAT1, H3K27ac, H3K9ac, and Nox5 proteins were colocalized in the area of CD45+/CD68+ immune cells and lipid-rich deposits within human atherosclerotic plaques. Lipopolysaccharide induced the levels of HAT1, H3K27ac, H3K9ac, and Nox5 and the recruitment of p300 and HAT1 at the sites of active transcription within Nox5 gene promoter in cultured human macrophages. Pharmacological inhibition of histone acetyltransferase significantly reduced the Nox5 gene and protein expression in lipopolysaccharide-challenged macrophages. The overexpression of p300 or HAT1 enhanced the Nox5 gene promoter activity. The histone acetyltransferase system is altered in human atherosclerosis. Under inflammatory conditions, HAT subtypes control Nox5 overexpression in cultured human macrophages. The data suggest the existence of a new epigenetic mechanism underlying oxidative stress in atherosclerosis.

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.

Human monocytes and macrophages express NADPH oxidase 5; a potential source of reactive oxygen species in atherosclerosis.

Monocytes (Mon) and Mon-derived macrophages (Mac) orchestrate important oxidative and inflammatory reactions in atherosclerosis by secreting reactive oxygen species (ROS) due, in large part, to the upregulated NADPH oxidases (Nox). The Nox enzymes have been extensively investigated in human Mon and Mac. However, the expression and functional significance of the Nox5 subtypes is not known. We aimed at elucidating whether Nox5 is expressed in human Mon and Mac, and examine its potential role in atherosclerosis. Human monocytic THP-1 cell line and CD14(+) Mon were employed to search for Nox5 expression. RT-PCR, Western blot, lucigenin-enhanced chemiluminescence and dihydroethidium assays were utilized to examine Nox5 in these cells. We found that Nox5 transcription variants and proteins are constitutively expressed in THP-1 cells and primary CD14(+) Mon. Silencing of Nox5 protein expression by siRNA reduced the Ca(2+)-dependent Nox activity and the formation of ROS in Mac induced by A23187, a selective Ca(2+) ionophore. Exposure of Mac to increasing concentrations of IFNγ (5-100 ng/ml) or oxidized LDL (5-100 µg/ml) resulted in a dose-dependent increase in Nox5 protein expression and elevation in intracellular Ca(2+) concentration. Immunohistochemical staining revealed that Nox5 is present in CD68(+) Mac-rich area within human carotid artery atherosclerotic plaques. To the best of our knowledge, this is the first evidence that Nox5 is constitutively expressed in human Mon. Induction of Nox5 expression in IFNγ- and oxidized LDL-exposed Mac and the presence of Nox5 in Mac-rich atheroma are indicative of the implication of Nox5 in atherogenesis.

High-glucose-increased expression and activation of NADPH oxidase in human vascular smooth muscle cells is mediated by 4-hydroxynonenal-activated PPARα and PPARß/δ.

High glucose induces vascular smooth muscle cell (SMC) dysfunction by generating oxidative stress attributable, in part, to the up-regulated NADPH oxidases (Nox). We have attempted to elucidate the high-glucose-generated molecular signals that mediate this effect and hypothesize that products of high-glucose-induced lipid peroxidation regulate Nox by activating peroxisome proliferator-activated receptors (PPARs). Human aortic SMCs were exposed to glucose (5.5-25 mM) or 4-hydroxynonenal (1-25 µM, 4-HNE). Lucigenin assay, real-time polymerase chain reaction, western blot, and promoter analyses were employed to investigate Nox. We found that high glucose generated an increase in Nox activity and expression. It also promoted oxidative stress that consequently induced lipid peroxidation, which resulted in the production of 4-HNE. Pharmacological inhibition of Nox activity significantly reduced the formation of high-glucose-induced 4-HNE. Exposure of SMCs to non-cytotoxic concentrations (1-10 µM) of 4-HNE alone mimicked the effect of high glucose incubation, whereas scavenging of 4-HNE by N-acetyl L-cysteine completely abolished both the effects of high glucose and 4-HNE. The latter exerted its effect by activating PPARα and PPARß/δ, but not PPARγ, as assessed pharmacologically by the inhibitory effect of selective antagonists and following the silencing of the expression of these receptors. These new data indicate that 4-HNE, generated following Nox activation, functions as an endogenous activator of PPARα and PPARß/δ. The newly discovered "lipid peroxidation products-PPARs-Nox axis" represents a novel mechanism of Nox regulation and an additional therapeutic target for oxidative stress in diabetes.

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/EBPß and C/EBPδ protein expression levels. Silencing of C/EBPα, C/EBPß 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/EBPß 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/EBPß 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.

Positive regulation of NADPH oxidase 5 by proinflammatory-related mechanisms in human aortic smooth muscle cells.

NADPH oxidase Nox5 subtype expression is significantly increased in vascular smooth muscle cells (SMCs) underlying fibro-lipid atherosclerotic lesions. The mechanisms that up-regulate Nox5 are not understood. Consequently, we characterized the promoter of the human Nox5 gene and investigated the role of various proinflammatory transcription factors in the regulation of Nox5 in human aortic SMCs. The Nox5 promoter was cloned in the pGL3 basic reporter vector. Functional analysis was done employing 5' deletion mutants to identify the sequences necessary to effect high levels of expression in SMCs. Transcriptional initiation site was detected by rapid amplification of the 5'-cDNA ends. In silico analysis indicated the existence of typical NF-kB, AP-1, and STAT1/STAT3 sites. Transient overexpression of p65/NF-kB, c-Jun/AP-1, or STAT1/STAT3 increased significantly the Nox5 promoter activity. Chromatin immunoprecipitation demonstrated the physical interaction of c-Jun/AP-1 and STAT1/STAT3 proteins with the Nox5 promoter. Lucigenin-enhanced chemiluminescence, real-time PCR, and Western blot assays showed that pharmacological inhibition and the silencing of p65/NF-kB, c-Jun/AP-1, or STAT1/STAT3 reduced significantly the interferon γ-induced Ca(2+)-dependent Nox activity and Nox5 expression. Up-regulated Nox5 correlated with increases in intracellular Ca(2+), an essential condition for Nox5 activity. NF-kB, AP-1, and STAT1/STAT3 are important regulators of Nox5 in SMCs by either direct or indirect mechanisms. Overexpressed Nox5 may generate free radicals in excess, further contributing to SMCs dysfunction in atherosclerosis.

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.

Transcriptional regulation of NADPH oxidase isoforms, Nox1 and Nox4, by nuclear factor-kappaB 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.

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.

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 gamma (IFNgamma) 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 IFNgamma-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)-alpha. 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-alpha-dependent upregulation of NADPHox activity and mRNA expression (NOX1, NOX4, p67(phox), p47(phox), p22(phox)). Inhibitors of AP-1 significantly diminished the Ang II or TNF-alpha-stimulated p22(phox) promoter activity and protein level. Transient overexpression of c-Jun/c-Fos upregulated p22(phox) 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 p22(phox) gene promoter. CONCLUSIONS: In SMCs exposed to Ang II or TNF-alpha, inhibition of AP-1-related pathways reduces NADPHox expression and the O(2)(-) production. The physical interaction of AP-1 with p22(phox) gene promoter facilitates NADPHox regulation.

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 Alzheimer's disease (AD) and Parkinson's 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.

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 p22(phox). RESULTS: The mRNA expression of p22(phox), p47(phox), p67(phox) and NOX 1 subunits, and the lack of gp91(phox) component, were detected in PCs by reverse transcriptase PCR. Western-blotting analysis demonstrated the protein expression of p22(phox), p47(phox) and p67(phox) subunits. As compared with the normal condition, stimulation of PCs with angiotensin II or high glucose induced: (i) an increase in ROS production and NAD(P)H oxidase activity, and (ii) an up-regulation of p22(phox) mRNA and protein expression. CONCLUSIONS: Taken together, the present study provides the first evidence that PCs express a functional phagocyte-type NAD(P)H oxidase, which is up-regulated by both angiotensin II and high glucose. Given the importance of ROS in vascular physiology and pathology, the NAD(P)H oxidase complex could be an important therapeutic target in the treatment of microvascular disorders.

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. A three 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.