Potentiation of nitric oxide-induced apoptosis in p53-/- vascular smooth muscle cells

The functionalrole of p53 in nitric oxide (NO)-mediated vascular smooth muscle cell(VSMC) apoptosis remains unknown. In this study, VSMC fromp53^/ and p53^+/+ murine aortas were exposedto exogenous or endogenous sources of NO. Unexpectedly,p53^/ VSMC were much more sensitive to theproapoptotic effects of NO than were p53^+/+ VSMC.Furthermore, this paradox appeared to be specific to NO, because otherproapoptotic agents did not demonstrate this differential effect onp53^/ cells. NO-induced apoptosis inp53^/ VSMC occurred independently of cGMP generation.However, mitogen-activated protein kinase (MAPK) pathways appeared toplay a significant role. Treatment of the p53^/ VSMCwith S-nitroso-N-acetylpenicillamine resulted ina marked activation of p38 MAPK and, to a lesser extent, of c-JunNH₂-terminal kinase, mitogen-activated protein kinasekinase (MEK) 1/2, and p42/44 (extracellular signal-regulated kinase,ERK). Furthermore, basal activity of the MEK-p42/44 (ERK)pathway was increased in the p53^+/+ VSMC. Inhibition of p38MAPK with SB-203580 or of MEK1/2 with PD-98059 blocked NO-inducedapoptosis. Therefore, p53 may protect VSMC against NO-mediatedapoptosis, in part, through differential regulation of MAPK pathways.

     

Ventilatory muscle activation and inflammation: cytokines, reactive oxygen species, and nitric oxide.

Strenuous diaphragmatic contractions that are induced by inspiratory resistive breathing initiate an inflammatory response that involves the elevation of pro- and anti-inflammatory cytokines within the diaphragm, which may then spill into the circulation. The production of reactive oxygen species within working respiratory muscles increases in response to these strenuous diaphragmatic contractions. At the same time, diaphragmatic nitric oxide (NO) production declines significantly, despite a time-dependent increase in NO synthase isoform protein expression. The increase in adhesion molecule expression and infiltration of granulocytes and macrophages that follows may contribute to the contraction-induced diaphragm injury. Enhanced generation of reactive oxygen species, oxidative stress augmentation, reduced NO production, and glycogen depletion are potential stimuli for the cytokine induction that is secondary to strenuous diaphragmatic contractions. This production of cytokines within the diaphragm may contribute to the diaphragmatic muscle fiber injury that occurs with strenuous contractions or to the expected repair process. TNF-alpha is a cytokine that compromises diaphragmatic contractility and may contribute to muscle wasting. IL-6 is a cytokine that may have beneficial systemic effects by mobilizing glucose from the liver and free fatty acids from the adipose tissue and providing them to the strenuously working respiratory muscles. Thus cytokine upregulation within the working diaphragm may be adaptive and maladaptive.     

Role of nitric oxide, reactive oxygen species, and p38 MAP kinase in the regulation of human chondrocyte apoptosis

This study addresses mechanisms by which interleukin-1beta (IL-1beta) regulates human chondrocyte apoptosis induced by a combination of the anti-CD95 antibody CH-11 and the proteasome inhibitor (PSI). The effect of IL-1beta on apoptosis varied among tissue samples. IL-1beta either enhanced (16/22 samples) or inhibited (6/22 samples) DNA fragmentation and caspase-3 processing. The protective effect of IL-1beta was abrogated by the nitric oxide (NO) synthesis inhibitor N-monomethyl-l-arginine (L-NMMA) while apoptosis stimulation was not affected. The NO-donors sodium nitroprusside (SNP) and S-nitroso-N-acetyl penicillamine (SNAP) blocked DNA fragmentation, and this was associated with partial inhibition of caspase-3 processing. Pyrrolidine dithiocarbamate (PDTC), a scavenger of reactive oxygen species (ROS) blocked apoptosis induction by CH-11/PSI as well as the enhancement by IL-1beta. The pro-apoptotic effects of IL-1beta were also abrogated by the p38 inhibitor SB 202190. In conclusion, IL-1beta augments CH-11/PSI induced apoptosis in the majority of chondrocyte samples. The pro-apoptotic effect of IL-1beta is not dependent on NO. In contrast, the anti-apoptotic effect of IL-1beta observed in a minority of samples is partially NO-dependent.     

Role of reactive oxygen species in bradykinin-induced proliferation of vascular smooth muscle cells

In addition to the induction of cell proliferation and migration, bradykinin (BK) can increase c-fos mRNA expression, activate ERK 1/2 and generate reactive oxygen species (ROS) in vascular smooth muscle cells (VSMC). It is not known, however, whether BK can induce cellular proliferation and extracellular matrix production via redox-sensitive signaling pathways. We investigated the role(s) of ROS in proliferation, migration and collagen synthesis induced by BK in VSMC derived from Sprague Dawley rat aorta. BK (10 nM) increased VSMC proliferation by 30% (n=5); this proliferation was inhibited by the antioxidants N-acetylcysteine (20 mM) and alpha-lipoic acid (LA, 250 mM). In addition, BK induced an increase in cell migration and in collagen levels that were blocked by LA. ROS production induced by BK (n=10) was significantly inhibited by bisindolylmaleimide (4microM) and by PD98059 (40microM). These results suggest that: 1) ROS participate in the mechanism(s) used by bradykinin to induce cellular proliferation; 2) bradykinin induces ROS generation through a pathway that involves the kinases PKC and MEK; and 3) ROS participate in the pathways mediating cell migration and the production of collagen as a response to treatment with bradykinin. To our knowledge, this is the first report describing mechanisms to explain the participation of ROS in the cellular proliferation and extracellular matrix pathway regulated by BK.     

LncRNA GAS5 regulates vascular smooth muscle cell cycle arrest and apoptosis via p53 pathway

Vascular remodeling is a pathological process following cardiovascular intervention. Vascular smooth muscle cells (VSMC) play a critical role in the vascular remodeling. Long noncoding RNAs (lncRNA) are a class of gene regulators functioning through various mechanisms in physiological and pathological conditions. By using cultured VSMC and rat carotid artery balloon injury model, we found that lncRNA growth arrest specific 5 (GAS5) serves as a negative regulator for VSMC survival in vascular remodeling. By manipulating GAS5 expression via adenoviral overexpression or short hairpin RNA knockdown, we found that GAS5 suppresses VSMC proliferation while promoting cell cycle arrest and inducing cell apoptosis. Mechanistically, GAS5 directly binds to p53 and p300, stabilizes p53-p300 interaction, and thus regulates VSMC cell survival via induction of p53-downstream target genes. Importantly, local delivery of GAS5 via adenoviral vector suppresses balloon injury-induced neointima formation along with an increased expression of p53 and apoptosis in neointimal SMCs. Our study demonstrated for the first time that GAS5 negatively impacts VSMC survival via activation the p53 pathway during vascular remodeling.     

GD3 recruits reactive oxygen species to induce cell proliferation and apoptosis in human aortic smooth muscle cells

Sialic acid containing glycosphingolipids (gangliosides) are expressed on the surface of all mammalian cells and have been implicated in regulating various biological phenomena; however, the detailed signaling mechanisms involved in this process are not known. We report here a novel aspect of disialoganglioside, GD3-mediated regulation of cell proliferation and cell death via the recruitment of reactive oxygen species (ROS). A low concentration (2.5-10 microm) of GD3, incubated with human aortic smooth muscle cells for a short period of time (10-30 min), stimulates superoxide generation via the activation of both NADPH oxidase and NADH oxidase activity. This leads to downstream signaling leading to cell proliferation and apoptosis. However, [(3)H]GD3 incubated with the cells under such conditions was found in a trypsin-sensitive fraction that was separable from endogenous GD3. The exact mechanism causing ROS generation and downstream signaling remains to be elucidated. The uptake of GD3 was accompanied by a 2.5-fold stimulation in the activity of mitogen-activated protein (MAP) kinase and 5-fold stimulation in cell proliferation. Preincubation of cells with membrane-permeable antioxidants, pyrrolidine dithiocarbamate, and N-acetylcysteine abrogated the superoxide generation and cell proliferation. In contrast, at higher concentrations (50-200 microm) GD3 inhibited the generation of superoxides but markedly stimulated the generation of nitric oxide (NO) (10-fold compared with control). This in turn stimulated mitochondrial cytochrome c release and intrachromosomal DNA fragmentation, which lead to apoptosis. In sum, at a low concentration, GD3 recruits superoxides to activate p44 MAPK and stimulates cell proliferation. In contrast, at high concentrations GD3 recruits nitric oxide to scavenge superoxide radicals that triggered signaling events that led to apoptosis. These observations might have relevance in regard to the potential role of GD3 in aortic smooth muscle cell proliferation and apoptosis that may contribute to plaque rupture in atherosclerosis.     

Interplay between ferritin metabolism, reactive oxygen species and nitric oxide

 The biological relevance of each of the three inorganic species – iron, oxygen, and nitric oxide (NO) – is crucial. Moreover, their metabolic pathways cross each other and thus create a complex network of connections responsible for the regulation of many essential biological processes. The iron storage protein ferritin, one of the main regulators of iron homeostasis, influences oxygen and NO metabolism. Here, examples are given of the biological interactions of the ferritin molecule (ferritin iron and ferritin shell) with reactive oxygen species (ROS) and NO. The focus is the regulation of ferritin expression by ROS and NO. From these data, ferritin emerges as an important cytoprotective component of the cellular response to ROS and NO. Also, by its ability to alter the amount of intracellular "free" iron, ferritin may affect the metabolism of ROS and NO. It is proposed that this putative activity of ferritin may constitute a missing link in the regulatory loop between iron, ROS, and NO. Received: 2 January 1997 / Accepted: 9 June 1997     

Role of reactive oxygen species and p53 in chromium(VI)-induced apoptosis

Apoptosis is a programmed cell death mechanism to control cell number in tissues and to eliminate individual cells that may lead to disease states. The present study investigates chromium(VI) (Cr(VI))-induced apoptosis and the role of reactive oxygen species (ROS) and p53 in this response. Treatment of human lung epithelial cells (A549) with Cr(VI) caused apoptosis as measured by DNA fragmentation, mitochondria damage, and cell morphology. Cr(VI)-induced apoptosis is contributed to ROS generation, resulting from cellular reduction of Cr(VI) as measured by flow cytometric analysis of the stained cells, oxygen consumption, and electron spin resonance spin trapping. Scavengers of ROS, such as catalase, aspirin, and N-acetyl-L-cysteine, decreased Cr(VI)-induced apoptosis, whereas NADPH and glutathione reductase, enhancers of Cr(VI)-induced ROS generation, increased it. p53 is activated by Cr(VI), mostly by ROS-mediated free radical reactions. Cr(VI)-induced ROS generation occurred within a few minutes after Cr(VI) treatment of the cells, whereas p53 induction took at least 5 h. The level of Cr(VI)-induced apoptosis was similar in both p53-positive cells and p53-negative cells independent of p53 status in the early stage (0-3 h) of Cr(VI) treatment. However, at the later stage (3-24 h), the level of the apoptosis is higher in p53-positive cells than in p53-negative cells. These results suggest that ROS generated through Cr(VI) reduction is responsible to the early stage of apoptosis, whereas p53 contributes to the late stage of apoptosis and is responsible for the enhancement of Cr(VI)-induced apoptosis at this stage.     

一氧化氮在17-β雌二醇抑制血管平滑肌细胞增殖和原癌基因c -fos表达中的作用

实验利用大鼠血管平滑肌细胞（vascular smooth muscle cells,VSMC)作为模型,观察17-β雌二醇（E2）对VSMC增殖和原癌基因c-fos表达的影响,并探讨VSMC源性一氧化氮（NO）在基中的作用,检测指标包括NO释放的测定,细胞计数、^3H-Tdr掺入,噻唑蓝（MTT）测定和c-fosmRNA表达,结果显示,E2（10^-12-10^-8mol/L)呈浓度依赖性地促进VSMC中NO的释放;10^-8mol/LE2能明显抑制10%小牛血清（FCS）和10^-7mol/L内皮素-1（ET-1）诱导的细胞增殖和DNA合成,E2的抑制作用均可被雌激素受体（ER）拮抗剂tamoxifen(10^-7mol/L)和一氧化氮合酶抑制剂L-NAME（10^-6mol/L)明显减轻;E2（10^-8mol/L)可明显抑制10^-7mol/LET-1诱导的VSMCc-fos表达,这种抑制作用可被L-NAME（10^-6mol/L)明显减轻,这些结果提示E2能抑制VSMC增殖和原癌基因c-fos表达,这种促进VSMC的NO释放密切相关,而且该作用至少部分通过ER介导。     

Influence of induced reactive oxygen species in p53-mediated cell fate decisions

The p53 tumor suppressor gene can induce either apoptosis or a permanent growth arrest (also termed senescence) phenotype in response to cellular stresses. We show that the increase in intracellular reactive oxygen species (ROS) associated with the magnitude of p53 protein expression correlated with the induction of either senescence or apoptosis in both normal and cancer cells. ROS inhibitors ameliorated both p53-dependent cell fates, implicating ROS accumulation as an effector in each case. The absence of Bax or PUMA strongly inhibited both p53-induced apoptosis and ROS increase, indicating an important role these p53 targets affecting mitochondrial function genes in p53-mediated ROS accumulation. Moreover, physiological p53 levels in combination with an exogenous ROS source were able to convert a p53 senescence response into apoptosis. All of these findings establish a critical role of ROS accumulation and mitochondrial function in p53-dependent cell fates and show that other ROS inducers can collaborate with p53 to influence these fate decisions. Thus, our studies imply that therapeutic agents that generate ROS are more likely to be toxic for normal cells than p53-negative tumor cells and provide a rationale for identifying therapeutic agents that do not complement p53 in ROS generation to ameliorate the cytotoxic side effects in normal cells.     

Important role for Rac1 in regulating reactive oxygen species generation and pulmonary arterial smooth muscle cell growth

Vascular NADPH oxidases have been shown to be a major source of reactive oxygen species (ROS). Recent studies have also implicated ROS in the proliferation of vascular smooth muscle cells. However, the components required for activation of the NADPH oxidase complex have not been clearly elucidated. Here we demonstrate that ROS generation in ovine pulmonary arterial smooth muscle cells (PASMCs) requires the activation of Rac1, implicating this protein as an important subunit of the NADPH oxidase complex. Our results, using a geranylgeranyl transferase inhibitor (GGTI-287), demonstrated a dose-dependent inhibition of Rac1 activity and ROS production. This was associated with an inhibition of PASMC proliferation with an arrest at G(2)/M. The inhibition of Rac1 by GGTI-287 led us to more specifically target Rac1 to investigate its role in the generation of ROS and cellular proliferation. To accomplish this, we utilized a dominant negative Rac1 (N17Rac1) and a constitutively active Rac1 (V12Rac1). These two forms of Rac1 were transiently expressed in PASMCs using adenovirus-mediated gene transfer. N17Rac1 expression resulted in decreased cellular Rac1 activity, whereas V12Rac1 infection showed increased activity. Compared with controls, the V12Rac1-expressing cells had higher levels of ROS production and increased proliferation, whereas the N17Rac1-expressing cells had decreased ROS generation and proliferation and cell cycle arrest at G(2)/M. However, the inhibition of cell growth produced by N17Rac1 overexpression could be overcome if cells were co-incubated with the Cu,Zn superoxide dismutase inhibitor DETC. These results indicate the importance of Rac1 in ROS generation and proliferation of vascular smooth muscle cells.     

一氧化氮在吲哚昔酚抑制血管平滑肌细胞增殖中的作用

目的：观察吲哚昔酚（ldoxifene,ldo）对大鼠血管平滑肌细胞增殖的影响,并探讨平滑肌源性一氧化氮（NO）在其中的作用。方法：血管平滑肌细胞培养、NO释放的测定、细胞计数和MTT测定。结果：吲哚昔酚可剂量依赖性的促使血管平滑肌细胞NO的释放,10μmol／L吲哚昔酚明显抑制10％胎牛血清（FCS）和10^-7mol／L的ET-1诱导的细胞增殖,吲哚昔酚的抑制作用可被一氧化氮合酶抑制剂L-NAME（100μmol／L）和鸟苷酸环化酶（guanylate cyclase,GC）抑制剂美蓝（methylene blue,MB）（10μmol／L）明显减轻。结论：吲哚昔酚抑制血管平滑肌细胞增殖的作用与其NO释放密切相关,其中可能有NO-GC-cGMP通路的参与。     

The role of nitric oxide,reactive oxygen species,and protein kinase C in oxytocin-induced cardioprotection in ischemic rat heart

Ischemia–reperfusion injury is a common complication of heart disease that is the leading cause of death worldwide. Here, we plan to elucidate oxytocin cardioprotection effects against ischemia–reperfusion via nitric oxide (NO), reactive oxygen species (ROS), and protein kinase C (PKC) in anesthetized rat preconditioned myocardium. Forty-eight Sprague-Dawley rats were equally divided into eight groups. All animals were subjected to 25 min ischemia and 120 min reperfusion. Oxytocin (OT), L-NAME (LNA, a nitric oxide synthase inhibitor), chelerythrine (CHE, a PKC enzyme inhibitor), and N-acetylcysteine (NAC, a ROS scavenger) were used prior to ischemia. Results showed that mean arterial pressure significantly reduced during the first 10 min of ischemia and reperfusion in IR, LNA, CHE, and NAC groups (p < 0.05). OT prevented mean arterial pressure decline during early phase of ischemia and reperfusion. Cardioprotective effects of OT in infarct size, plasma levels of creatine kinase-MB and lactate dehydrogenase, severity and incidence of ventricular arrhythmias were abolished by L-NAME, chelerythrine, and N-acetylcysteine (p < 0.05). The present study showed that OT pretreatment reduces myocardial infarct size and ventricular arrhythmias, and improves mean arterial pressure via NO production, PKC activation, and ROS balance. These findings provide new insight into therapeutic strategies for ischemic heart disease.     

PLD1 promotes reactive oxygen species production in vascular smooth muscle cells and injury-induced neointima formation

Phospholipase D (PLD) generates the signaling lipid phosphatidic acid (PA) and has been known to mediate proliferation signal in vascular smooth muscle cells (VSMCs). However, it remains unclear how PLD contributes to vascular diseases. VSMC proliferation directly contributes to the development and progression of cardiovascular disease, such as atherosclerosis and restenosis after angioplasty. Using the mouse carotid artery ligation model, we find that deletion of Pld1 gene inhibits neointima formation of the injuried blood vessels. PLD1 deficiency reduces the proliferation of VSMCs in both injured artery and primary cultures through the inhibition of ERK1/2 and AKT signals. Immunohistochemical staining of injured artery and flow cytometry analysis of VSMCs shows a reduction of the levels of reactive oxygen species (ROS) in Pld1^?/? VSMCs. An increase of intracellular ROS by hydrogen peroxide stimulation restored the reduced activities of ERK and AKT in Pld1^?/? VSMCs, whereas a reduction of ROS by N-acetyl-l-cysteine (NAC) scavenger lowered their activity in wild-type VSMCs. These results indicate that PLD1 plays a critical role in neointima, and that PLD1 mediates VSMC proliferation signal through promoting the production of ROS. Therefore, inhibition of PLD1 may be used as a therapeutic approach to suppress neointimal formation in atherosclerosis and restenosis after angioplasty.     

Methylglyoxal-induced nitric oxide and peroxynitrite production in vascular smooth muscle cells

Methylglyoxal (MG) is a metabolite of glucose. Our previous study demonstrated an elevated MG level with an increased oxidative stress in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats. Whether MG causes the generation of nitric oxide (NO) and superoxide anion (O2*-), leading to peroxynitrite (ONOO-) formation in VSMCs, was investigated in the present study. Cultured rat thoracic aortic SMCs (A-10) were treated with MG or other different agents. Oxidized DCF, reflecting H2O2 and ONOO- production, was significantly increased in a concentration- and time-dependent manner after the treatment of SMCs with MG (3-300 microM) for 45 min-18 h (n = 12). MG-increased oxidized DCF was effectively blocked by reduced glutathione or N-acetyl-l-cysteine, as well as L-NAME (p < 0.05, n = 12). Both O2*- scavenger SOD and NAD(P)H oxidase inhibitor DPI significantly decreased MG-induced oxidized DCF formation. MG significantly and concentration-dependently increased NO and O2*- generation in A-10 cells, which was significantly inhibited by L-NAME and SOD or DPI, respectively. In conclusion, MG induces significant generation of NO and O2*- in rat VSMCs, which in turn causes ONOO- formation. An elevated MG level and the consequential ROS/RNS generation would alter cellular signaling pathways, contributing to the development of different insulin resistance states such as diabetes or hypertension.     

Induction of nitric oxide synthase by cytokines in vascular smooth muscle cells.

We investigated the mechanisms by which cytokines lead to a diminished responsiveness of vascular smooth muscle to vasoconstrictors. The attenuation of noradrenaline-induced contraction by 6 to 24 h incubations with the cytokines, tumor necrosis factor and interleukin-1, in endothelium-denuded rabbit aorta was associated with an increase in intracellular cyclic GMP level. This increase was abolished by the stereoselective inhibitor of nitric oxide-synthase, NG-nitro-L-arginine and by cycloheximide. Formation of nitric oxide was detected in the cytosol of cytokine-treated native and cultured smooth muscle cells by activation of purified soluble guanylate cyclase, and depended on tetrahydrobiopterin, but not on Ca2(+)-calmodulin. The results indicate that cytokines induce a nitric oxide-synthase of the macrophage-type in vascular smooth muscle.     

Protective effect of p53 in vascular smooth muscle cells against nitric oxide-induced apoptosis is mediated by up-regulation of heme oxygenase-2

The tumor suppressor gene p53 regulates apoptotic cell death and the cell cycle. In this study, we investigated the role of p53 in nitric oxide (NO)-induced apoptosis in vascular smooth muscle cells (VSMCs). We found that the NO donor S-nitroso-N-acetylpenicillamine (SNAP) increased apoptotic cell death in p53-deficient VSMCs compared with wild-type cells. The heme oxygenase (HO) inhibitor tin protoporphyrin IX reduced the resistance of wild-type VSMCs to SNAP-induced cell death. SNAP promoted HO-1 expression in both cell types. HO-2 protein was increased only in wild-type VSMCs following SNAP treatment; however, similar levels of HO-2 mRNA were detected in both cell types. SNAP significantly increased the levels of non-heme-iron and dinitrosyl iron-sulfur clusters in wild-type VSMCs compared with p53-deficient VSMCs. Moreover, pretreatment with FeSO4 and the carbon monoxide donor CORM-2, but not biliverdin, significantly protected p53-deficient cells from SNAP-induced cell death compared with normal cells. These results suggest that wild-type VSMCs are more resistant to NO-mediated apoptosis than p53-deficient VSMCs through p53-dependent up-regulation of HO-2.     

Release of nitric oxide from human vascular smooth muscle cells

It has recently been shown that nitric oxide (NO) or a labile NO-releasing compound is produced in endothelial cells. In the present study we measured the NO-release from human vascular smooth muscle cells in culture. The cells released an average 2.2 x 10(-9) moles nitric oxide per 10(8) cells in ten minutes with a large variation between different cell lines and passages without stimulators. The NO-release was markedly reduced by the inhibitor of NO-formation NG-momomethyl-L-arginine (L-NMMA, 10(-5) M) to 3% of the control levels (p less than 0.02, n = 4), but unaffected by acetylcholine, bradykinin or endothelin -1, -2 or -3. In a microperfusion system the compound released from human vascular smooth muscle cells caused profound relaxation of isolated rat mesenteric resistance arteries. Thus, human vascular smooth muscle cells in culture produce and release biologically active NO from L-arginine.