ET-1 stimulates pulmonary arterial smooth muscle cell proliferation via induction of reactive oxygen species

Recent studies implicate reactive oxygen species (ROS) such as superoxide anions and H(2)O(2) in the proliferation of systemic vascular smooth muscle cells (SMCs). However, the role of ROS in SMC proliferation within the pulmonary circulation remains unclear. We investigated the effects of endothelin-1 (ET-1), a potential SMC mitogen, on ROS production and proliferation of fetal pulmonary artery SMCs (FPASMCs). Exposure to ET-1 resulted in increases in superoxide production and viable FPASMCs after 72 h. These increases were prevented by pretreatment with PD-156707. Treatment with pertussis toxin blocked the effects of ET-1, whereas cholera toxin stimulated superoxide production and increased viable cell numbers even in the absence of ET-1. Wortmannin, LY-294002, diphenyleneiodonium (DPI), 4-(2-aminoethyl)benzenesulfonyl fluoride, and apocynin also prevented the ET-1-mediated increases in superoxide production and viable cell numbers. Exposure to H(2)O(2) or diethyldithiocarbamate increased viable cell number by 37% and 50%, respectively. Conversely, ascorbic acid and DPI decreased viable cell number, which appeared to be due to an increase in programmed cell death. Our data suggest that ET-1 exerts a mitogenic effect on FPASMCs via an increase in ROS production and that antioxidants can block this effect via induction of apoptosis. Antioxidant treatment may therefore represent a potential therapy for pulmonary vascular diseases.     

Reactive oxygen-mediated contraction in pulmonary arterial smooth muscle: cellular mechanisms.

Reactive oxygen species (at least relatively high doses) cause contraction of pulmonary arterial smooth muscle. The objective of the present study was to elucidate the possible cellular mechanisms involved in reactive oxygen-mediated contraction. Isolated arterial rings from Sprague-Dawley rats were placed in tissue baths containing Earle's balanced salt solution. The maximum active force production (Po) in response to 80 mM KCl was obtained. All other responses were normalized as percentages of Po for comparative purposes. Exposure to reactive oxygen (generated from either the xanthine oxidase reaction (XO) or the glucose oxidase reaction) resulted in pulmonary arterial muscle developing mean active tension of 17.1 +/- 3.0% Po. This contraction was independent of extracellular calcium, since it was not affected by verapamil (a calcium channel blocker) or by placement of the arterial muscle in calcium-free media. Phentolamine (an alpha 1-receptor blocker) and propranolol (a beta-receptor blocker) did not diminish the response to XO. Ryanodine (a SR calcium release inhibitor), while reducing the response to norepinephrine, did not affect the response to XO. However, H-7 (an inhibitor of protein kinase C) decreased the XO-mediated contraction by 49%. These results indicate that while Ca2+ may not be involved as a second messenger, protein kinase C activity appears to play a role in the transduction pathway of reactive oxygen species mediated contraction of pulmonary arterial smooth muscle.     

Reactive oxygen species alter autocrine and paracrine signaling

Cytochrome P450 (P450) 3A4 (CYP3A4) is the most abundant P450 protein in human liver and intestine and is highly inducible by a variety of drugs and other compounds. The P450 catalytic cycle is known to uncouple and release reactive oxygen species (ROS), but the effects of ROS from P450 and other enzymes in the endoplasmic reticulum have been poorly studied from the perspective of effects on cell biology. In this study, we expressed low levels of CYP3A4 in HepG2 cells, a human hepatocarcinoma cell line, and examined effects on intracellular levels of ROS and on the secretion of a variety of growth factors that are important in extracellular communication. Using the redox-sensitive dye RedoxSensor red, we demonstrate that CYP3A4 expression increases levels of ROS in viable cells. A custom ELISA microarray platform was employed to demonstrate that expression of CYP3A4 increased secretion of amphiregulin, intracellular adhesion molecule 1, matrix metalloprotease 2, platelet-derived growth factor (PDGF), and vascular endothelial growth factor, but suppressed secretion of CD14. The antioxidant N-acetylcysteine suppressed all P450-dependent changes in protein secretion except for CD14. Quantitative RT-PCR demonstrated that changes in protein secretion were consistently associated with corresponding changes in gene expression. Inhibition of the NF-κB pathway blocked P450 effects on PDGF secretion. CYP3A4 expression also altered protein secretion in human mammary epithelial cells and C10 mouse lung cells. Overall, these results suggest that increased ROS production in the endoplasmic reticulum alters the secretion of proteins that have key roles in paracrine and autocrine signaling.     

Reactive oxygen species from smooth muscle mitochondria initiate cold-induced constriction of cutaneous arteries

Cold constricts cutaneous blood vessels by selectively increasing the activity of smooth muscle alpha2-adrenoceptors (alpha2-ARs). In mouse tail arteries, alpha2-AR constriction is mediated by alpha2A-ARs at 37 degrees C, whereas the cold-induced augmentation in alpha2-AR activity is mediated entirely by alpha2C-ARs. Cold causes translocation of alpha2C-ARs from the trans-Golgi to the plasma membrane, mediated by cold-induced activation of RhoA and Rho kinase. The present experiments analyzed the mechanisms underlying these responses. Mouse tail arteries were studied in a pressure myograph. Cooling the arteries (28 degrees C) caused a rapid increase in reactive oxygen species (ROS) in smooth muscle cells, determined by confocal microscopy of arteries loaded with the ROS-sensitive probes, dichlorodihydrofluorescein or reduced Mitotracker Red. The inhibitor of mitochondrial complex I rotenone (10 micromol/l), the antioxidant N-acetylcysteine (NAC; 20 mmol/l), or the cell-permeable mimic of superoxide dismutase MnTMPyP (50 micromol/l) did not affect vasoconstriction to alpha2-AR stimulation (UK-14304) at 37 degrees C but dramatically inhibited the response at 28 degrees C. Indeed, these ROS inhibitors abolished the cold-induced increase in alpha2-AR constrictor activity. NAC (20 mmol/l) or MnTMPyP (50 micromol/l) also abolished the cold-induced activation of RhoA in human cultured vascular smooth muscle cells and the cold-induced mobilization of alpha2C-ARs to the cell surface in human embryonic kidney 293 cells transfected with the receptor. The combined results suggest that cold-induced constriction is mediated by redox signaling in smooth muscle cells, initiated by mitochondrial generation of ROS, which stimulate RhoA/Rho kinase signaling and the subsequent mobilization of alpha2C-ARs to the cell surface. Altered activity of ROS may contribute to cold-induced vasospasm occurring in Raynaud's phenomenon.     

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.     

Reactive oxygen species sensitivity of angiotensin II-dependent translation initiation in vascular smooth muscle cells

Translation initiation, the rate-limiting step in protein synthesis, is a key event in vascular smooth muscle cell growth, a major component of vascular disease. Translation initiation is regulated by interaction between PHAS-I and the eukaryotic initiation factor 4E (eIF4E). Although angiotensin II (Ang II)-induced vascular smooth muscle cell hypertrophy requires the generation of reactive oxygen species (ROS), the ROS sensitivity of these events and their upstream activators remain unclear. Here, we investigated the role of ROS in the regulation of PHAS-I phosphorylation on Thr-70 and Ser-65, an event required for the release of eIF4E from PHAS-I. Ang II-induced Ser-65 phosphorylation was ROS-dependent as assessed by pretreatment with ebselen (3.6 +/- 0.2 versus 1.1 +/- 0.2), diphenylene iodonium (3.6 +/- 0.2 versus 1.0 +/- 0.1), and N-acetyl cysteine (3.6 +/- 0.2 versus 1.2 +/- 0.1), but Ang II-stimulated phosphorylation of Thr-70 was ROS-insensitive. Although phosphatidylinositol 3-kinase pathway inhibition by LY294004 blocked both Ser-65 and Thr-70 phosphorylation (3.8 +/- 0.1 versus 0.8 +/- 0.1 and 3.2 +/- 0.2 versus 1.0 +/- 0.01, respectively), protein phosphatase 2A inhibition by okadaic acid selectively increased (3.3 +/- 0.1 versus 5.2 +/- 0.1) and p38 mitogen-activated protein kinase inhibition by SB203580 selectively decreased (3.8 +/- 0.1 versus 1.4 +/- 0.3) Ser-65 phosphorylation. Dominant negative Akt adenovirus also inhibited only Ser-65 phosphorylation (3.7 +/- 0.1 versus 1.0 +/- 0.03). These results demonstrate a unique differential ROS sensitivity of two separate residues on PHAS-I, which seems to be explained by the selective involvement of distinct signaling pathways in the regulation of these phosphorylation events.     

Reactive oxygen species mediate RhoA/Rho kinase-induced Ca2+ sensitization in pulmonary vascular smooth muscle following chronic hypoxia

Recent evidence supports a prominent role for Rho kinase (ROK)-mediated pulmonary vasoconstriction in the development and maintenance of chronic hypoxia (CH)-induced pulmonary hypertension. Endothelin (ET)-1 contributes to the pulmonary hypertensive response to CH, and recent studies by our laboratory and others indicate that pulmonary vascular reactivity following CH is largely independent of changes in vascular smooth muscle (VSM) intracellular free calcium concentration ([Ca(2+)](i)). In addition, CH increases generation of reactive oxygen species (ROS) in pulmonary arteries, which may underlie the shift toward ROK-dependent Ca(2+) sensitization. Therefore, we hypothesized that ROS-dependent RhoA/ROK signaling mediates ET-1-induced Ca(2+) sensitization in pulmonary VSM following CH. To test this hypothesis, we determined the effect of pharmacological inhibitors of ROK, myosin light chain kinase (MLCK), tyrosine kinase (TK), and PKC on ET-1-induced vasoconstriction in endothelium-denuded, Ca(2+)-permeabilized small pulmonary arteries from control and CH (4 wk at 0.5 atm) rats. Further experiments examined ET-1-mediated, ROK-dependent phosphorylation of the regulatory subunit of myosin light chain phosphatase (MLCP), MYPT1. Finally, we measured ET-1-induced ROS generation in dihydroethidium-loaded small pulmonary arteries and investigated the role of ROS in mediating ET-1-induced, RhoA/ROK-dependent Ca(2+) sensitization using the superoxide anion scavenger, tiron. We found that CH increases ET-1-induced Ca(2+) sensitization that is sensitive to inhibition of ROK and MLCK, but not PKC or TK, and correlates with ROK-dependent MYPT1(Thr696) phosphorylation. Furthermore, tiron inhibited basal and ET-1-stimulated ROS generation, RhoA activation, and VSM Ca(2+) sensitization following CH. We conclude that CH augments ET-1-induced Ca(2+) sensitization through ROS-dependent activation of RhoA/ROK signaling in pulmonary VSM.     

Mechanical plasticity and contractile properties of airway smooth muscle

Smooth muscle has the unique ability to adapt easily and quickly to length changes without compromising its ability to generate force. This ability is referred to as mechanical plasticity and is now considered to be an important aspect of smooth muscle that affects both its contractile and relaxation behaviour. It is therefore important to incorporate knowledge of plasticity into further studies of smooth muscle behaviour. It is also important that future studies be focused on deciphering the mechanism of smooth muscle length adaptation and plasticity. This review outlines some of the proposed mechanisms determining plasticity. However, it should be said that there are other proposed mechanisms not touched upon here, which may be equally as important. This review also focuses on the relevance of smooth muscle plasticity in asthma, but it is important to remember that there are other places where smooth muscle plasticity may play an equally important role.     

Reactive oxygen species in pulmonary inflammation by ambient particulates

Exposure to ambient air pollution particles (PM) has been associated with increased cardiopulmonary morbidity and mortality, particularly in individuals with pre-existing disease. Exacerbation of pulmonary inflammation in susceptible people (e.g., asthmatics, COPD patients) appears to be a central mechanism by which PM exert their toxicity. Health effects are seen most consistently with PM with aerodynamic diameter < 2.5 micrometers (PM(2.5)), although 10 micrometers < PM < 2.5 micrometers can also be toxic. Through its metal, semi-quinone, lipopolysaccaride, hydrocarbon, and ultrafine constituents, PM may exert oxidative stress on cells in the lung by presenting or by stimulating the cells to produce reactive oxygen (ROS). In vivo, PM increase cytokine and chemokine release, lung injury, and neutrophil influx. In vitro analysis of PM effects on the critical cellular targets, alveolar macrophages, epithelial cells, and neutrophils, demonstrates PM- and oxidant-dependent responses consistent with in vivo data. These effects have been observed with PM samples collected over years as well as concentrated PM(2.5) (CAPs) collected in real time. Oxidative stress mediated by ROS is an important mechanism of PM-induced lung inflammation.     

Dexamethasone induces apoptosis in pulmonary arterial smooth muscle cells

Background

Dexamethasone suppressed inflammation and haemodynamic changes in an animal model of pulmonary arterial hypertension (PAH). A major target for dexamethasone actions is NF-κB, which is activated in pulmonary vascular cells and perivascular inflammatory cells in PAH. Reverse remodelling is an important concept in PAH disease therapy, and further to its anti-proliferative effects, we sought to explore whether dexamethasone augments pulmonary arterial smooth muscle cell (PASMC) apoptosis.

Methods

Analysis of apoptosis markers (caspase 3, in-situ DNA fragmentation) and NF-κB (p65 and phospho-IKK-α/β) activation was performed on lung tissue from rats with monocrotaline (MCT)-induced pulmonary hypertension (PH), before and after day 14–28 treatment with dexamethasone (5 mg/kg/day). PASMC were cultured from this rat PH model and from normal human lung following lung cancer surgery. Following stimulation with TNF-α (10 ng/ml), the effects of dexamethasone (10⁻⁸–10⁻⁶ M) and IKK2 (NF-κB) inhibition (AS602868, 0–3 μM (0-3×10⁻⁶ M) on IL-6 and CXCL8 release and apoptosis was determined by ELISA and by Hoechst staining. NF-κB activation was measured by TransAm assay.

Results

Dexamethasone treatment of rats with MCT-induced PH in vivo led to PASMC apoptosis as displayed by increased caspase 3 expression and DNA fragmentation. A similar effect was seen in vitro using TNF-α-simulated human and rat PASMC following both dexamethasone and IKK2 inhibition. Increased apoptosis was associated with a reduction in NF-κB activation and in IL-6 and CXCL8 release from PASMC.

Conclusions

Dexamethasone exerted reverse-remodelling effects by augmenting apoptosis and reversing inflammation in PASMC possibly via inhibition of NF-κB. Future PAH therapies may involve targeting these important inflammatory pathways.     

低氧条件下大鼠肺动脉平滑肌细胞中活性氧与低氧诱导因子-1α和细胞增殖的关系

在低氧条件下,观察大鼠肺动脉平滑肌细胞（pulmonary arterial smooth muscle cells,PASMCs）中活性氧（reactive oxygen species,ROS）的变化,探讨ROS的变化是否通过调控低氧诱导因子-4α（hypoxia-inducible factor 1α, HIF-1α）的表达影响PASMCs的增殖。采用组织块法原代培养大鼠PASMCs,分成3组：常氧组（21％O2,24h）,低氧组（5％O2,24h）,低氧＋Mn-TBAP组（5％O2,24h,Mn-TBAP是一种ROS清除剂）。用激光共聚焦显微镜荧光染色法检测细胞内ROS的变化;用RT-PCR和免疫组织化学方法分别测定HIF-1α mRNA和蛋白的表达;用MTT法检测细胞增殖程度。结果显示：（1）低氧组PASMCs内ROS水平明显高于常氧组（P〈0．05）,低氧＋Mn-TBAP组ROS水平明显低于低氧组（P〈0．05）,但仍高于常氧组（P〈0．05）;（2）低氧组及低氧＋Mn-TBAP组的HIF-1α mRNA和蛋白表达均高于常氧组（P〈0．05）,且低氧组表达高于低氧＋Mn-TBAP组（P〈0．05）;（3）低氧组细胞增殖明显高于常氧组和低氧＋Mn-TBAP组（P〈0．05）,低氧＋Mn-TBAP组细胞增殖高于常氧组（P〈0．05）。结果表明：在低氧条件下大鼠PASMCs中ROS水平明显升高,RROS的变化能够调节HIF-1α的表达,进而影响平滑肌细胞的增殖,提示ROS可能在肺动脉高压的发病机制和低氧信号转导中具有重要作用。     

Effect of noradrenaline on the electrical and contractile properties of smooth muscle cells in the pulmonary artery]

Experiments were performed on the smooth muscle cells of rabbit a. pulmonalis using the microelectrode technique. No spontaneous electrical or mechanical activity was recorded in normal Krebs solution. The current-voltage relation in these smooth muscle cells showed marked rectification. No changes in the isometric tension were observed due to the anodal or cathodal stimulating currents. Strong depolarization of the muscle cells produced only local potentials on the cathelectrotone which never developed into a spike. Noradrenaline (10(-8) g/ml) caused depolarization of the 5-7 mV in the muscle cell membrane and a considerable contraction of the muscle strip as well. Under such conditions the contractile apparatus of the muscle cells became sensible to the resting potential level. Anodal stimulation was accompanied by relaxation of the muscle strip, whereas cathodal stimulation--by its contraction. The alpha-adrenoblocking agent (phentolamine) blocked the effect of noradrenaline evidencing the fact that noradrenaline exerted its excitatory action on the smooth muscle cells of the a. pulmonalis through the alpha-adrenoreceptors.     

Vascular smooth muscle contractile properties in the turtle Pseudemys scripta elegans

1. Turtle aortic rings were characterized by high frequency spontaneous contractile activity and variable responsiveness to constrictor agents.2. The tissue response was remarkably insensitive to temperature at a range of 37°–15°C.3. The contractile response was effectively blocked by the calcium channel antagonist nifedipine and was substantially dependent on extracellular calcium concentrations.4. Lowering the sodium concentration of the bath medium resulted in a strong, transient contraction followed by reduced responsiveness to norepinephrine and the absence of spontaneous activity.5. Disruption of the vessel endothelium resulted in enhanced and reduced responsiveness to norepinephrine (NE) and acetylcholine (ACh), respectively.6. The results indicate that the regulation of contractile function in turtle vascular smooth muscle differs in several respects from that of mammalian tissue, perhaps, reflecting the adaptation of the vasculature to low pressure and ectothermic conditions.     

Reactive oxygen species and ERK 1/2 mediate monocyte chemotactic protein-1-stimulated smooth muscle cell migration

Summary Monocyte chemotactic protein-1 (MCP-1), a potent chemoattractant for monocytes, is thought to play a major role in atherosclerosis, but whether its atherogenic effects involve the direct modulation of vascular smooth muscle cell (SMC) functions remains unclear. This study examined the effects of MCP-1 on the migration of cultured A7r5 SMCs and the signaling pathways involved. Addition of recombinant MCP-1 stimulated SMC migration in modified Boyden chambers coated with type I collagen in a concentration-dependent manner, with 10^–9 M being maximally effective. Using untreated A7r5 cells, two MCP-1 receptors, CCR2 and CCR4, were detected and MCP-1 secretion was significantly increased by stimulation with platelet-derived growth factor. MCP-1-stimulated A7r5 migration was completely blocked by the NAD(P)H oxidase inhibitor, diphenylene iodonium (DPI), and dose-dependently inhibited by polyethylene glycol-conjugated superoxide dismutase (PEG-SOD), suggesting a role for reactive oxygen species (ROS) in this process. During MCP-1 stimulation, ROS production increased rapidly, then gradually decayed over 60 min, and this effect was markedly decreased by pretreatment with DPI or PEG-SOD. Interestingly, U0126 and PD98059, which inhibit activation of extracellular signal-regulated kinases 1/2 (ERK 1/2), significantly inhibited MCP-1-activated ROS generation. Furthermore, transfection of an active mutant of MEK1 (ERK 1/2 kinase) markedly increased superoxide production in rat aortic smooth muscle cells, as detected by dihydroethydium staining, suggesting that ERK 1/2 activation stimulates ROS generation. ERK 1/2 activation was increased for at least 30 min in cells incubated with MCP-1, and this effect was abolished by U0126 or DPI pretreatment. These results demonstrate that MCP-1 is a chemoattractant for SMCs and that MCP-1-stimulated migration requires both ROS production and ERK 1/2 activation in a positive activation loop, which may contribute to the atherogenic effects of MCP-1.These authors contributed equally to this work.