Oxidative Stress In Vascular Cells Research Articles

Antioxidants modulate the antiproliferative effects of nitric oxide on vascular smooth muscle cells and adventitial fibroblasts by regulating oxidative stress

Background S-nitrosothiols (SNO) release nitric oxide (NO) through interaction with ascorbic acid (AA). However, little is known about their combined effect in the vasculature. The aim of this study was to investigate the effect of AA on SNO-mediated NO release, proliferation, cell cycle progression, cell death, and oxidative stress in vascular cells. Methods Vascular smooth muscle cells and adventitial fibroblasts harvested from the aortae of Sprague–Dawley rats were treated with AA, ± S-nitrosoglutathione (GSNO), or ± diethylenetriamine NONOate (DETA/NO). NO release, proliferation, cell cycle progression, cell death, and oxidative stress were determined by the Griess reaction, [ 3H]-thymidine incorporation, flow cytometry, trypan blue exclusion, and 5-(and-6)chloromethyl-2′,7′dichlorodihydrofluorescein staining, respectively. Results AA increased NO release from GSNO 3-fold ( P < .001). GSNO and DETA/NO significantly decreased proliferation, but AA abrogated this effect ( P < .05). Mirroring the proliferation data, changes in cell cycle progression induced by GSNO and DETA/NO were reversed by the addition of AA. GSNO- and DETA/NO-mediated increases in oxidative stress were significantly decreased by the addition of AA ( P < .001). Conclusions Despite causing increased NO release from GSNO, AA reduced the antiproliferative and cell cycle effects of GSNO and DETA/NO through the modulation of oxidative stress.

Evidence for Increased Methylglyoxal in the Vasculature of Women With Preeclampsia

Preeclampsia is characterized by vascular endothelial dysfunction partly attributed to oxidative stress. In the vasculature of preeclamptic women, we have shown increased lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1) and arginase expression, which can contribute to vascular oxidative stress. However, the mechanisms of such upregulation are unknown. Methylglyoxal (MG) that plays a role in the vascular complications of diabetes mellitus and the development of hypertension can be one potential factor that can affect LOX-1 and arginase through its ability to induce oxidative stress in vascular cells. MG also reacts with lysine residues in proteins to generate advanced glycation end product, N(epsilon)-carboxy ethyl lysine, which also serves as a marker of MG. We hypothesized that markers of MG formation will be increased in the vasculature of preeclamptic women and that exogenous MG will induce oxidative stress by the upregulation of LOX-1 via arginase. We observed increased N(epsilon)-carboxy ethyl lysine expression in the vasculature of women with preeclampsia in comparison with normotensive pregnant women. Moreover, glyoxalase I and II, enzymes that detoxify MG, and glutathione reductase, which generates reduced glutathione, a cofactor for glyoxalase, are also reduced in preeclampsia. In cultured endothelial cells, MG increased arginase expression by 6 hours and LOX-1 expression by 24 hours. Inhibition of arginase or NO synthase significantly reduced MG-induced LOX-1 expression, superoxide levels, and nitrotyrosine staining. In conclusion, MG-induced LOX-1 expression is mediated via arginase upregulation likely because of uncoupling of NO synthase, which may have implications in preeclampsia.

Angiotensin II triggers release of leukotriene C4 in vascular smooth muscle cells via the multidrug resistance-related protein 1

The multidrug resistance-related protein-1 (MRP1) is important for the management of oxidative stress in vascular cells in vivo. Substrates of MRP1 are, among others, glutathione and the leukotriene C(4) (LTC(4)), an eicosanoid and mediator of inflammation. Angiotensin (Ang) II infusion results in MRP1(-/-) mice compared to wild-type mice in improved endothelial function and reduced reactive oxygen species (ROS) formation. However, the interaction between Ang II, LTC(4) and MRP1 is not completely understood and has never been investigated in vitro. Ang II induced in vascular smooth muscle cells (VSMC) the release of LTC(4) and the generation of ROS. Pharmacologic inhibition of MRP1 via MK 571 significantly reduced Ang II-induced ROS release (L012-luminescence) in VSMC. The release of ROS after Ang II stimulation is inhibited, to a comparable degree, by blockade of the Cys-LT1 receptor with montelukast. Incubation of VSMC with recombined LTC(4) and Ang II caused enhanced rates of proliferation in VSMC. This effect can be rescued by either MRP1 or Cys-LT1 receptor inhibition. Accordingly, stimulation of VSMC with LTC(4) reduces intracellular levels of glutathione, but does not affect apoptosis. LTC(4) stimulation results in a significant activation of MRP1, but does not alter MRP1 expression. These findings indicate a connection between Ang II, MRP1 and LTC(4). Both, MRP1 and LTC(4), are potentially promising targets for atheroprotective therapy.

Multidrug Resistance Protein-1 Affects Oxidative Stress, Endothelial Dysfunction, and Atherogenesis via Leukotriene C 4 Export

We recently showed that the multidrug resistance related protein-1 (MRP1) is important for the management of oxidative stress in vascular cells. However, the underlying mechanism and the in vivo relevance of these findings remain elusive. We hypothesize that inside-outside transport of leukotriene C(4) (LTC(4)) via MRP1 is a substantial proatherogenic mechanism in the vasculature. To test this hypothesis, we investigated the effects of MRP1 inhibition and LTC(4) receptor blockade (Cys-LT1 receptor) in vitro and in vivo. MRP1 is expressed abundantly in vascular smooth muscle cells (VSMCs). Pharmacological inhibition of MRP1 via MK571 reduces angiotensin II-induced reactive oxygen species release by 59% (L012 fluorescence) in VSMCs. The release of reactive oxygen species after angiotensin II stimulation also is inhibited by blockade of the Cys-LT1 receptor with montelukast. Incubation of VSMCs with recombined LTC(4) causes enhanced rates of reactive oxygen species and proliferation in wild-type and MRP1(-/-) VSMCs. Accordingly, the LTC(4) release in the cell culture supernatant of MRP1(-/-) VSMCs is significantly decreased compared with wild-type cells. To extend our observations to the in vivo situation, atherosclerosis-prone apolipoprotein E-deficient mice on a high-cholesterol diet were treated with placebo, the MRP1 inhibitor MK571, or the Cys-LT1 receptor inhibitor montelukast for 6 weeks. Treatment with MK571 or montelukast reduced vascular reactive oxygen species production, significantly improved endothelial function, and ameliorated atherosclerotic plaque generation by 52% and 61%, respectively. These findings indicate that MRP1 and LTC(4) exert proatherosclerotic effects and that both MRP1 and LTC(4) are potentially promising targets for atheroprotective therapy.

Paraoxonase-2 Reduces Oxidative Stress in Vascular Cells and Decreases Endoplasmic Reticulum Stress–Induced Caspase Activation

In the vascular system, elevated levels of reactive oxygen species (ROS) produce oxidative stress and predispose to the development of atherosclerosis. Therefore, it is important to understand the systems producing and those scavenging vascular ROS. Here, we analyzed the ROS-reducing capability of paraoxonase-2 (PON2) in different vascular cells and its involvement in the endoplasmic reticulum stress pathway known as the unfolded protein response. Quantitative real-time polymerase chain reaction and Western blotting revealed that PON2 is equally expressed in vascular cells and appears in 2 distinct glycosylated isoforms. By determining intracellular ROS, we show that overexpression of PON2 markedly reduced ROS, whereas its knockdown increased ROS levels significantly. Using microscopic and biochemical methods, we found PON2 mainly in the nuclear membrane and endoplasmic reticulum. Furthermore, PON2 expression was induced at both the promoter and protein levels by endoplasmic reticulum stress pathway unfolded protein response. This pathway may promote both apoptotic and survival mechanisms. Functionally, PON2 reduced unfolded protein response-accompanying oxidative stress and unfolded protein response-derived caspase activation. We suggest that PON2 represents an endogenous defense mechanism against vascular oxidative stress and unfolded protein response-induced cell death, thereby contributing to the prevention of atherosclerosis.

Effects of PGC-1α on TNF-α–Induced MCP-1 and VCAM-1 Expression and NF-κB Activation in Human Aortic Smooth Muscle and Endothelial Cells

Increased oxidative stress in vascular cells is implicated in the pathogenesis of atherosclerosis. Reactive oxygen species (ROS) induce vascular inflammation via the proinflammatory cytokine/NF-kappaB pathway. Several lines of evidence suggest that peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC-1alpha) is an important regulator of intracellular ROS levels. However, no studies have examined the effects of PGC-1alpha on this process. We investigated the effects of PGC-1alpha on inflammatory molecule expression and activity of the redox-sensitive transcription factor, NF-kappaB, in vascular cells. PGC-1alpha expressed in human aortic smooth (HASMCs) and endothelial cells (HAECs) is upregulated by AMP-activated protein kinase activators, including metformin, rosiglitazone and alpha-lipoic acid. Tumor necrosis factor-alpha (TNF-alpha), a major proinflammatory factor in the development of vascular inflammation, stimulates intracellular ROS production through an increase in both mitochondrial ROS and NAD(P)H oxidase activity. Adenovirus-mediated overexpression of the PGC-1alpha gene in HASMCs and HAECs leads to a significant reduction in intracellular and mitochondrial ROS production as well as NAD(P)H oxidase activity. Consequently, NF-kappaB activity and MCP-1 and VCAM-1 induced by TNF-alpha are suppressed. Our data support the possibility that agents stimulating PGC-1alpha expression in the vasculature aid in preventing the development of atherosclerosis.

Homocysteine stimulates phosphorylation of NADPH oxidase p47phoxand p67phoxsubunits in monocytes via protein kinase Cβ activation

Hyperhomocysteinaemia is an independent risk factor for cardiovascular diseases due to atherosclerosis. The development of atherosclerosis involves reactive oxygen species-induced oxidative stress in vascular cells. Our previous study [Wang and O (2001) Biochem. J. 357, 233-240] demonstrated that Hcy (homocysteine) treatment caused a significant elevation of intracellular superoxide anion, leading to increased expression of chemokine receptor in monocytes. NADPH oxidase is primarily responsible for superoxide anion production in monocytes. In the present study, we investigated the molecular mechanism of Hcy-induced superoxide anion production in monocytes. Hcy treatment (20-100 microM) caused an activation of NADPH oxidase and an increase in the superoxide anion level in monocytes (THP-1, a human monocytic cell line). Transfection of cells with p47phox siRNA (small interfering RNA) abolished Hcy-induced superoxide anion production, indicating the involvement of NADPH oxidase. Hcy treatment resulted in phosphorylation and subsequently membrane translocation of p47phox and p67phox subunits leading to NADPH oxidase activation. Pretreatment of cells with PKC (protein kinase C) inhibitors Ro-32-0432 (bisindolylmaleimide XI hydrochloride) (selective for PKCalpha, PKCbeta and PKCgamma) abolished Hcy-induced phosphorylation of p47phox and p67phox subunits in monocytes. Transfection of cells with antisense PKCbeta oligonucleotide, but not antisense PKCalpha oligonucleotide, completely blocked Hcy-induced phosphorylation of p47phox and p67phox subunits as well as superoxide anion production. Pretreatment of cells with LY333531, a PKCbeta inhibitor, abolished Hcy-induced superoxide anion production. Taken together, these results indicate that Hcy-stimulated superoxide anion production in monocytes is regulated through PKC-dependent phosphorylation of p47phox and p67phox subunits of NADPH oxidase. Increased superoxide anion production via NADPH oxidase may play an important role in Hcy-induced inflammatory response during atherogenesis.

Endothelial NO Synthase Target of Aldosterone

The mineralocorticoid hormone aldosterone plays a pivotal role in sodium resorption and potassium excretion and, consequently, in fluid balance and blood pressure homeostasis.1 Current studies indicate that mineralocorticoid receptors are present not only in epithelial cells, and aldosterone also acts on nonepithelial tissues, including the heart, blood vessels, and brain. In the vascular system, mineralocorticoid receptors and the enzyme 11-β-hydroxy-steroid dehydrogenase type 2, which confers aldosterone specificity to the mineralocorticoid receptor, could be localized in endothelial and vascular smooth muscle cells.2 Other than the mineralocorticoid receptor, steroidogenic enzymes necessary to synthesize aldosterone are also expressed in extra-adrenal tissues, including the vascular wall, which, although still controversial, may be consistent with de novo aldosterone production acting in an autocrine/paracrine fashion. Aldosterone induces oxidative stress in vascular cells through NADPH oxidase activation, which plays a central role for endothelial dysfunction and atherosclerotic vascular disease.3 Mineralocorticoid receptor blockade increased NO bioavailability and improved impaired endothelial function by decreasing oxidative stress in hypertension, atherosclerosis, and heart failure.3–5 In a rat model of heart failure, aldosterone antagonism reduced vascular superoxide anion formation, and in combination with an angiotensin-converting enzyme inhibitor, increased the expression of the endothelial NO synthase (eNOS) and restored the …

Protein disulfide isomerase is a central regulator of NADPH oxidase activity

Redox state is a critical factor determining enzyme activity. The protein disulfide isomerase (PDI) belongs to a class of enzymes that maintains proteins in the adequate redox state by isomerising thiols. Vascular NADPH oxidases are important sources of reactive oxygen species (ROS). As NADPH oxidases are likely to be thiol oxidized by the radicals generated by themselves, we hypothesize that the PDI promotes oxidative stress in vascular cells by maintaining NADPH oxidase activity. Overexpression of fluorescence-tagged fusion constructs revealed colocalization of the NADPH oxidase proteins Nox1, Nox2 and Nox4 with endogenous PDI. Moreover, immuno-precipitation of PDI resulted in co-precipitation of Nox 1, 2 and 4 as well as of the p22phox subunit of the NAPDH oxidase. ROS formation in NADPH oxidase-over-expressing HEK cells was reduced by siRNA directed against PDI. Analysis of NADPH oxidase homologues revealed several highly conserved cysteines. Exchange of some of these amino acids to valine or serine resulted in a substantial loss of function. In cultured vascular smooth muscle cells (VSMC) inhibition of PDI using bacitracin or DNA antisense oligonucleotides inhibited NADPH oxidase activity and significantly attenuated angiotensin II-induced radical-mediated activation of AKT. Therefore, PDI via its action on redox-sensitive cysteines is a novel regulator of NADPH oxidases.

Effects of Recombinant Adenovirus-Mediated Uncoupling Protein 2 Overexpression on Endothelial Function and Apoptosis

Increased oxidative stress in vascular cells plays a key role in the development of endothelial dysfunction and atherosclerosis. Uncoupling protein 2 (UCP2) is an important regulator of intracellular reactive oxygen species (ROS) production. This study was undertaken to test the hypothesis that, UCP2 functions as an inhibitor of the atherosclerotic process in endothelial cells. Adenovirus-mediated UCP2 (Ad-UCP2) overexpression led to a significant increase in endothelial nitric oxide synthase (eNOS) and decrease in endothelin-1 mRNA expression in human aortic endothelial cells (HAECs). Moreover, UCP2 inhibited the increase in ROS production and NF-kappaB activation, and apoptosis of HAECs induced by lysophophatidylcholine (LPC) and linoleic acid. LPC and linoleic acid caused mitochondrial calcium accumulation and transient mitochondrial membrane hyperpolarization, which was followed by depolarization. UCP2 overexpression prevented these processes. In isolated rat aorta, Ad-UCP2 infection markedly improved impaired vascular relaxation induced by LPC. The data collectively suggest that UCP2, functions as a physiologic regulator of ROS generation in endothelial cells. Thus, measures to increase UCP2 expression in vascular endothelial cells may aid in preventing the development and progression of atherosclerosis in patients with metabolic syndrome.

Angiotensin II produces superoxide-mediated impairment of endothelial function in cerebral arterioles.

Angiotensin II (Ang II) produces oxidative stress in vascular cells in culture and in extracranial conduit arteries. The goal of this study was to examine the hypothesis that Ang II produces superoxide-mediated impairment of endothelial function in cerebral microvessels. Diameter of cerebral arterioles (baseline diameter=104+/-3 microm) was measured with the use of a closed cranial window in anesthetized rabbits. Topical application of Ang II was used to avoid effects on arterial pressure. Ang II (0.1 to 1 micromol/L for 2 hours) had no effect on baseline diameter (change in diameter of -3+/-2% in response to 1 micromol/L Ang II) but produced concentration-dependent inhibition of vasodilatation to the endothelium-dependent agonist bradykinin. For example, 1 micromol/L Ang II inhibited responses to 1 nmol/L bradykinin by almost 80%. These inhibitory effects of Ang II were prevented by the superoxide scavenger 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron; 10 mmol/L) or diphenylene iodonium (DPI; 3 micromol/L), an inhibitor of NAD(P)H oxidase. Ang II did not inhibit vasodilatation in response to nitroprusside, an endothelium-independent vasodilator. These findings are the first evidence that local Ang II produces superoxide-mediated vascular dysfunction in cerebral microvessels. The results with DPI suggest that the source of superoxide may be an NAD(P)H oxidase.