Neutralization of Acidic Industrial Wastes and Fixation of Trace Element by Oil Shale Ash: Formation of a Green Product.

Oil shale is a rock that contains organic matter in a concentration that allows it to be used as an energy source. As a result of the shale combustion process, large amounts of two types of ash are formed: fly ash (∼10%) and bottom ash (∼90%). At present, in Israel, only fly oil shale ash is used, which constitutes a minority of the oil shale burn products, whereas bottom oil shale ash is accumulated as waste. Bottom ash contains a high percentage of calcium in the form of anhydrite (CaSO4) and calcite (CaCO3). Thus, it can be used to neutralize acidic waste and to fix trace elements. This study examined the process of scrubbing the acid waste by the ash, its characterization pre- and post-upgrade treatment, to test its suitability as a partial substitute for aggregates, natural sand, and cement in concrete mixtures. In the current study, we compared the chemical and physical characterization of oil shale bottom ash before and after upgrading the ash via chemical treatment. In addition, its utilization as a scrubbing reagent for acidic wastes from the phosphate industry was studied.

TGA-DSC Combined Coal Analysis as a Tool for QC (Quality Control) and Reactivity Patterns of Coals.

Coal is at present a major fuel source for power generation worldwide and will remain as such in the near future. The most important property of coal that determines its price is its calorific value. However, volatiles, ash, and moisture content are also very important properties needed for the quality control (QC) of the coal used to maintain an optimal operation of coal combustion in a boiler. The determination of these properties is carried out via well-established ASTM/DIN methods, which are slow and time-consuming. This study uses combined thermogravimetric analysis (TGA)/differential thermal calorimetry (DSC) instrumentation as a tool to evaluate the reactivity patterns of the aliphatic versus aromatic content of coals, which is correlated to the volatile content of coals. Two coals, bituminous (American Baily Pittsburgh No. 6) used in Israeli utilities and lignite (brown coal Hambach) used in German power plants, have been investigated in this study. The results show that the combined TG/DSC method can provide a much better understanding of the chemical reactivity of coals in the combustion process.

Mechanism Underlying the Emission of Gases during the Low-Temperature Oxidation of Bituminous and Lignite Coal Piles: The Involvement of Radicals.

Coal is one of the major fuels for power generation, and it will continue in this capacity for the next several decades. Two types of coal are mainly used: lignite and bituminous coals. When exposed to air, post-mining, the coal surface undergoes LTO (low-temperature oxidation) at RT-150 °C according to the atmospheric oxygen level. The LTO process decreases the calorific value of the coal, and consequently, different gases are released [mainly carbon oxides (CO, CO2), water vapor, hydrogen (H2), and also some low molecular-weight organic gases (C1-5)]. Some of these gases are toxic and flammable. In extreme cases, fires erupt. The mechanism by which the molecular oxygen oxidizes the coal macromolecule at the temperature range of 30-150 °C (LTO process) is complex and also involves a chain of radical reactions that take place; however, the exact underlying mechanism is not yet clear. The LTO process was studied in detail by simulating the processes occurring in the coal piles by using two coal types: an American Bailey coal, used in Israeli coal-fired utilities and a German Hambach lignite, used in German utilities. The mechanism underlying the LTO process and the radical reactions that are involved are discussed in detail.

Distribution of shortest cycle lengths in random networks.

We present analytical results for the distribution of shortest cycle lengths (DSCL) in random networks. The approach is based on the relation between the DSCL and the distribution of shortest path lengths (DSPL). We apply this approach to configuration model networks, for which analytical results for the DSPL were obtained before. We first calculate the fraction of nodes in the network which reside on at least one cycle. Conditioning on being on a cycle, we provide the DSCL over ensembles of configuration model networks with degree distributions which follow a Poisson distribution (Erdos-Rényi network), degenerate distribution (random regular graph), and a power-law distribution (scale-free network). The mean and variance of the DSCL are calculated. The analytical results are found to be in very good agreement with the results of computer simulations.

Mechanisms related to NO-induced motility in differentiated rat aortic smooth muscle cells.

Nitric oxide (NO) is thought to play an important role as an inhibitor of vascular cell proliferation, motility, and neointima formation. This effect is mediated, in part, via the upregulation of protein tyrosine phosphatase (PTP)1B. Conversely, studies have reported that in presumably hyperinsulinemic mice fed a high-fat diet, NO enhances vascular remodeling, whereas a deficit of NO attenuates vascular remodeling. We have reported that in differentiated cultured smooth muscle cells treated with insulin, NO induces a motogenic effect that is dependent on Src homology-2 domain PTP 2 (SHP2) upregulation. In the present study, we describe novel mechanisms relevant to the motogenic effect of NO. Treatment of cultured cells with the selective angiontensin type 1 receptor antagonist losartan, but not with the selective angiotensin type 2 receptor antagonist PD-123319, blocked the comotogenic capacity of NO and insulin. Insulin and NO increased the secretion of ANG II into the culture media by 2- and 2.5-fold (P < 0.05), respectively, whereas treatment of cells with ANG II uncovered the motogenic effect of NO (1.4-fold above control, P < 0.05) and decreased the levels of PTP1B to 45% of control (P < 0.05). Suppression of PTP1B function was sufficient to uncover the motogenic effect of NO. The capacity of insulin to suppress PTP1B activity was blocked by losartan, implicating ANG II function in mediating this effect. Both insulin and ANG II induced the upregulation of phosphatidyl inositol 3-kinase (PI3K)-δ by two- to threefold (P < 0.05), and this effect was both necessary and sufficient to uncover NO-induced motogenesis. Finally, suppression of PTP1B function potentiated, whereas overexpression of PTP1B inhibited, SHP2-induced motogenesis. These results support the hypothesis that the comotogenic effect of insulin and NO occurs via an ANG II-mediated effect involving the suppression of PTP1B and upregulation of PI3K-δ and SHP2.

Suppression of PKG by PDGF or nitric oxide in differentiated aortic smooth muscle cells: obligatory role of protein tyrosine phosphatase 1B.

Treatment of aortic smooth muscle cells with PDGF induces the upregulation of protein tyrosine phosphatase 1B (PTP1B). PTP1B, in turn, decreases the function of several growth factor receptors, thus completing a negative feedback loop. Studies have reported that PDGF induces the downregulation of PKG as part of a repertoire of dedifferentiation of vascular smooth muscle cells. Other studies have reported that chronic nitric oxide (NO) treatment also induces the downregulation of PKG. In the present study, we tested the hypothesis that the downregulation of PKG by PDGF or NO in differentiated rat aortic smooth muscle cells can be attributed to the upregulation of PTP1B. We found that treatment with PDGF or NO induced an upregulation of PTP1B levels. Overexpression of PTP1B induced a marked downregulation of PKG mRNA and protein levels, whereas the expression of dominant negative PTP1B or short interfering RNA directed against PTP1B blocked the capacity of PDGF or NO to decrease PKG levels. We conclude that the upregulation of PTP1B by PDGF or NO is both necessary and sufficient to induce the downregulation of PKG via an effect on PKG mRNA levels.

Chronic insulin treatment suppresses PTP1B function, induces increased PDGF signaling, and amplifies neointima formation in the balloon-injured rat artery.

We tested the hypothesis that hyperinsulinemia induces the suppression of protein tyrosine phosphatase 1B (PTP1B) function, leading to enhanced PDGF receptor (PDGFR) signaling and neointimal hyperplasia. Rats were implanted with insulin-releasing pellets or sham pellets. Blood glucose levels, insulin levels, food and water intake, body weights, and blood pressures were measured. Neointimal hyperplasia was assessed by computerized morphometry 14 days after carotid balloon injury. PTP1B protein expression in injured arteries was determined via Western blot analysis, whereas PTP1B activity was determined via an immunophosphatase assay. Serum insulin levels were two- to threefold greater in hyperinsulinemic rats, whereas systolic blood pressures, food and water intake, serum triglyceride levels, plasma cortisol levels, and urinary catecholamine levels were not affected. Fourteen days after injury, neointima-to-media area ratios were 0.89 +/- 0.23 and 1.35 +/- 0.22 in control and hyperinsulinemic rats, respectively (P < 0.01). PTP1B protein levels and total PTP1B activity in injured carotid arteries from the insulin-treated group were significantly decreased 7 or 14 days after injury, whereas PTP1B specific activity was decreased only 14 days after injury. These findings were associated with decreased PTP1B mRNA levels and increased PDGFR tyrosyl phosphorylation in insulin-treated rats. These observations support the hypothesis that hyperinsulinemia induces the suppression of PTP1B function, leading to enhanced PDGFR signaling and neointimal hyperplasia.

Cardiac fibrogenesis following infarction in mice with deletion of inducible nitric oxide synthase.

BACKGROUND: Studies have shown that the absence of inducible nitric oxide synthase (iNOS) improves cardiac function and survival after myocardial infarction (MI). The responsible mechanisms, however, remain uncertain. Cardiac iNOS is significantly increased after MI, which is colocalized with fibrous tissue formation. Herein, we tested our hypothesis that iNOS is involved in the development of cardiac fibrosis. METHODS: Wild-type and iNOS-knockout mice were subjected to MI by left coronary artery ligation. At week 1, 2, 3, and 4 post-MI, we addressed cardiac expression of profibrogenic mediator, growth of collagen-producing cells, collagen synthesis, and degradation. RESULTS: In the infarcted myocardium of wild-type and iNOS-knockout mice, transforming growth factor (TGF)-beta1 expression was significantly increased, particularly in the early stage; myofibroblasts appeared and became abundant for over 4 weeks; matrix metalloproteinase-1 expression was low, whereas tissue inhibitor of matrix metalloproteinase-1 was significantly elevated; type-I collagen mRNA was significantly increased and collagen was continuously accumulated. In the noninfarcted myocardium, TGF-beta1 and type-I collagen mRNA levels as well as collagen volume were also elevated, but less evident than infarcted myocardium. However, there was no significant difference in cardiac TGF-beta1 expression, myofibroblast population, collagen synthesis/degradation, and collagen volume between wild-type and iNOS-knockout mice with MI. CONCLUSION: The current study suggests that iNOS-induced nitric oxide production may not mediate cardiac fibrosis after MI. Thus, other mechanisms are involved in nitrosative stress-induced cardiac dysfunction after MI.

Chronic insulin treatment amplifies PDGF-induced motility in differentiated aortic smooth muscle cells by suppressing the expression and function of PTP1B.

Hyperinsulinemia plays a major role in the pathogenesis of vascular disease. Restenosis occurs at an accelerated rate in hyperinsulinemia and is dependent on increased vascular smooth muscle cell movement from media to neointima. PDGF plays a critical role in mediating neointima formation in models of vascular injury. We have reported that PDGF increases the levels of protein tyrosine phosphatase PTP1B and that PTP1B suppresses PDGF-induced motility in cultured cells and that it attenuates neointima formation in injured carotid arteries. Others have reported that insulin enhances the mitogenic and motogenic effects of PDGF in cultured smooth muscle cells and that hyperinsulinemia promotes vascular remodeling. In the present study, we tested the hypothesis that insulin amplifies PDGF-induced cell motility by suppressing the expression and function of PTP1B. We found that chronic but not acute treatment of cells with insulin enhances PDGF-induced motility in differentiated cultured primary rat aortic smooth muscle cells and that it suppresses PDGF-induced upregulation of PTP1B protein. Moreover, insulin suppresses PDGF-induced upregulation of PTP1B mRNA levels, PTP1B enzyme activity, and binding of PTP1B to the PDGF receptor-beta, and it enhances PDGF-induced PDGF receptor phosphotyrosylation. Treatment with insulin induces time-dependent upregulation of phosphatidylinositol 3-kinase (PI3-kinase)-delta and activation of Akt, an enzyme downstream of PI3-kinase. Finally, inhibition of PI3-kinase activity, or its function, by pharmacological or genetic means rescues PTP1B activity in insulin-treated cells. These observations uncover novel mechanisms that explain how insulin amplifies the motogenic capacity of the pivotal growth factor PDGF.

IP3 constricts cerebral arteries via IP3 receptor-mediated TRPC3 channel activation and independently of sarcoplasmic reticulum Ca2+ release.

Vasoconstrictors that bind to phospholipase C-coupled receptors elevate inositol-1,4,5-trisphosphate (IP(3)). IP(3) is generally considered to elevate intracellular Ca(2+) concentration ([Ca(2+)](i)) in arterial myocytes and induce vasoconstriction via a single mechanism: by activating sarcoplasmic reticulum (SR)-localized IP(3) receptors, leading to intracellular Ca(2+) release. We show that IP(3) also stimulates vasoconstriction via a SR Ca(2+) release-independent mechanism. In isolated cerebral artery myocytes and arteries in which SR Ca(2+) was depleted to abolish Ca(2+) release (measured using D1ER, a fluorescence resonance energy transfer-based SR Ca(2+) indicator), IP(3) activated 15 pS sarcolemmal cation channels, generated a whole-cell cation current (I(Cat)) caused by Na(+) influx, induced membrane depolarization, elevated [Ca(2+)](i), and stimulated vasoconstriction. The IP(3)-induced I(Cat) and [Ca(2+)](i) elevation were attenuated by cation channel (Gd(3+), 2-APB) and IP(3) receptor (xestospongin C, heparin, 2-APB) blockers. TRPC3 (canonical transient receptor potential 3) channel knockdown with short hairpin RNA and diltiazem and nimodipine, voltage-dependent Ca(2+) channel blockers, reduced the SR Ca(2+) release-independent, IP(3)-induced [Ca(2+)](i) elevation and vasoconstriction. In pressurized arteries, SR Ca(2+) depletion did not alter IP(3)-induced constriction at 20 mm Hg but reduced IP(3)-induced constriction by approximately 39% at 60 mm Hg. [Ca(2+)](i) elevations and constrictions induced by endothelin-1, a phospholipase C-coupled receptor agonist, were both attenuated by TRPC3 knockdown and xestospongin C in SR Ca(2+)-depleted arteries. In summary, we describe a novel mechanism of IP(3)-induced vasoconstriction that does not occur as a result of SR Ca(2+) release but because of IP(3) receptor-dependent I(Cat) activation that requires TRPC3 channels. The resulting membrane depolarization activates voltage-dependent Ca(2+) channels, leading to a myocyte [Ca(2+)](i) elevation, and vasoconstriction.

High tidal volume mechanical ventilation with hyperoxia alters alveolar type II cell adhesion.

Patients with acute respiratory distress syndrome undergoing mechanical ventilation may be exposed to both high levels of stretch and high levels of oxygen. We hypothesized that the combination of high stretch and hyperoxia promotes loss of epithelial adhesion and impairs epithelial repair mechanisms necessary for restoration of barrier function. We utilized a model of high tidal volume mechanical ventilation (25 ml/kg) with hyperoxia (50% O(2)) in rats to investigate alveolar type II (AT2) cell adhesion and focal adhesion signaling. AT2 cells isolated from rats exposed to hyperoxia and high tidal volume mechanical ventilation (MVHO) exhibited significantly decreased cell adhesion and reduction in phosphotyrosyl levels of focal adhesion kinase (FAK) and paxillin compared with control rats, rats exposed to hyperoxia without ventilation (HO), or rats ventilated with normoxia (MV). MV alone increased phosphorylation of p130(Cas). RhoA activation was increased by MV, HO, and the combination of MV and HO. Treatment of MVHO cells with keratinocyte growth factor (KGF) for 1 h upon isolation reduced RhoA activity and restored attachment to control levels. Attachment and migration of control AT2 cells was significantly decreased by constitutively active RhoA or a kinase inactive form of FAK (FRNK), whereas expression of dominant negative RhoA in cells from MVHO-treated rats restored cell adhesion. Mechanical ventilation with hyperoxia promotes changes in focal adhesion proteins and RhoA in AT2 cells that may be deleterious for cell adhesion and migration.

cAMP inhibits transforming growth factor-beta-stimulated collagen synthesis via inhibition of extracellular signal-regulated kinase 1/2 and Smad signaling in cardiac fibroblasts.

Cardiac fibroblasts produce and degrade extracellular matrix and are critical in regulating cardiac remodeling and hypertrophy. Cytokines such as transforming growth factor-beta (TGF-beta) play a fundamental role in the development of tissue fibrosis by stimulating matrix deposition and other profibrotic responses, but less is known about pathways that might inhibit fibrosis. Increased cAMP formation inhibits myofibroblast differentiation and collagen production by cardiac fibroblasts, but the mechanism of this inhibition is not known. We sought to characterize the signaling pathways by which cAMP-elevating agents alter collagen expression and myofibroblast differentiation. Treatment with 10 microM forskolin or isoproterenol increased cAMP production and cAMP response element binding protein (CREB) phosphorylation in cardiac fibroblasts and inhibited serum- or TGF-beta-stimulated collagen synthesis by 37% or more. These same cAMP-elevating agents blunted TGF-beta-stimulated expression of collagen I, collagen III, and alpha-smooth muscle actin. Forskolin or isoproterenol treatment blocked the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) induced by TGF-beta despite the fact that these cAMP-elevating agents stimulated ERK1/2 activation on their own. cAMP-elevating agents also attenuated the activation of c-Jun NH(2)-terminal kinase and reduced binding of the transcriptional coactivator CREB-binding protein 1 to transcriptional complexes containing Smad2, Smad3, and Smad4. Pharmacological inhibition of ERK completely blocked TGF-beta-stimulated collagen gene expression, but expression of an active mutant of MEK was additive with TGF-beta treatment. Thus, cAMP-elevating agents inhibit the profibrotic effects of TGF-beta in cardiac fibroblasts largely through inhibiting ERK1/2 phosphorylation but also by reducing Smad-mediated recruitment of transcriptional coactivators.

Role of protein-tyrosine phosphatase SHP2 in focal adhesion kinase down-regulation during neutrophil cathepsin G-induced cardiomyocytes anoikis.

Inflammatory cells and their proteases contribute to tissue reparation at site of inflammation. Although beneficial at early stages, excessive inflammatory reaction leads to cell death and tissue damage. Cathepsin G (Cat.G), a neutrophil-derived serine protease, has been shown to induce neonatal rat cardiomyocyte detachment and apoptosis by anoikis through caspase-3 dependent pathway. However the early mechanisms that trigger Cat.G-induced caspase-3 activation are not known. This study identifies focal adhesion kinase (FAK) tyrosine dephosphorylation as an early mechanism that regulates Cat.G-induced anoikis in cardiomyocytes. Both FAK tyrosine phosphorylation at Tyr-397 and kinase activity decrease rapidly upon Cat.G treatment and was associated with a decrease of FAK association with adapter and cytoskeletal proteins, p130(Cas) and paxillin, respectively. FAK-decreased tyrosine phosphorylation is required for Cat.G-induced myocyte anoikis as concurrent expression of phosphorylation-deficient FAK mutated at Tyr-397 or pretreatment with a protein-tyrosine phosphatase (PTP) inhibitor, pervanadate, blocks Cat.G-induced FAK tyrosine dephosphorylation, caspase-3 activation and DNA fragmentation. Analysis of PTPs activation shows that Cat.G treatment induces an increase of SHP2 and PTEN phosphorylation; however, only SHP2 forms a complex with FAK in response to Cat.G. Expression of dominant negative SHP2 mutant markedly attenuates FAK tyrosine dephosphorylation induced by Cat.G and protects myocytes to undergo apoptosis. In contrast, increased SHP2 expression exacerbates Cat.G-induced FAK tyrosine dephosphorylation and myocyte apoptosis. Taken together, these results show that Cat.G induces SHP2 activation that leads to FAK tyrosine dephosphorylation and promotes cardiomyocyte anoikis.

Counter-regulatory function of protein tyrosine phosphatase 1B in platelet-derived growth factor- or fibroblast growth factor-induced motility and proliferation of cultured smooth muscle cells and in neointima formation.

OBJECTIVE: We have previously reported that vascular injury or treatment of cultured vascular smooth muscle cells with platelet-derived growth factor-BB (PDGF-BB) or fibroblast growth factor-2 (FGF2) increases the levels of protein tyrosine phosphatase (PTP)1B. The current study was designed to test the hypothesis that PTP1B attenuates PDGF- or FGF-induced motility and proliferation of cultured cells, as well as neointima formation in injured rat carotid arteries. METHODS AND RESULTS: Treatment of cultured cells with adenovirus expressing PTP1B decreased PDGF-BB- or FGF2-induced cell motility and blocked PDGF-BB- or FGF2-induced proliferation, whereas expression of dominant negative PTP1B (C215S-PTP1B) uncovered the motogenic effect of subthreshold levels of PDGF-BB or FGF2, increased neointimal and medial cell proliferation, and induced neointimal enlargement after balloon injury. The inhibitory effect of PTP1B directed against PDGF in cultured cells was associated with dephosphorylation of the PDGFbeta receptor. CONCLUSIONS: PTP1B suppresses cell proliferation and motility in cultured smooth muscle cells treated with PDGF-BB or FGF2, and the phosphatase plays a counter-regulatory role in vascular injury-induced cell proliferation and neointima formation. Taken together with previous studies indicating increased PTP1B levels in cells treated with growth factors, the current findings are the first to report the existence of an inhibitory feedback loop involving PDGF or FGF, and PTP1B in blood vessels.

Nitric oxide attenuates IGF-I-induced aortic smooth muscle cell motility by decreasing Rac1 activity: essential role of PTP-PEST and p130cas.

Recent data support the hypothesis that reactive oxygen species (ROS) play a central role in the initiation and progression of vascular diseases. An important vasoprotective function related to the regulation of ROS levels appears to be the antioxidant capacity of nitric oxide (NO). We previously reported that treatment with NO decreases phosphotyrosine levels of adapter protein p130(cas) by increasing protein tyrosine phosphatase-proline, glutamate, serine, and threonine sequence protein (PTP-PEST) activity, which leads to the suppression of agonist-induced H(2)O(2) elevation and motility in cultured rat aortic smooth muscle cells (SMCs). The present study was performed to investigate the hypotheses that 1) IGF-I increases the activity of the small GTPase Rac1 as well as H(2)O(2) levels and 2) NO suppresses IGF-I-induced H(2)O(2) elevation by decreasing Rac1 activity via increased PTP-PEST activity and dephosphorylation of p130(cas). We report that IGF-I induces phosphorylation of p130(cas) and activation of Rac1 and that NO attenuates these effects. The effects of NO are mimicked by the overexpression of PTP-PEST or dominant-negative (dn)-p130(cas) and antagonized by the expression of dn-PTP-PEST or p130(cas). We conclude that IGF-I induces rat aortic SMC motility by increasing phosphotyrosine levels of p130(cas) and activating Rac1 and that NO decreases motility by activating PTP-PEST, inducing dephosphorylating p130(cas), and decreasing Rac1 activity. Decreased Rac1 activity lowers intracellular H(2)O(2) levels, thus attenuating cell motility.

Gab1, SHP2, and protein kinase A are crucial for the activation of the endothelial NO synthase by fluid shear stress.

Fluid shear stress enhances NO production in endothelial cells by a mechanism involving the activation of the phosphatidylinositol 3-kinase and the phosphorylation of the endothelial NO synthase (eNOS). We investigated the role of the scaffolding protein Gab1 and the tyrosine phosphatase SHP2 in this signal transduction cascade in cultured and native endothelial cells. Fluid shear stress elicited the phosphorylation and activation of Akt and eNOS as well as the tyrosine phosphorylation of Gab1 and its association with the p85 subunit of phosphatidylinositol 3-kinase and SHP2. Overexpression of a Gab1 mutant lacking the pleckstrin homology domain abrogated the shear stress-induced phosphorylation of Akt but failed to affect the phosphorylation or activity of eNOS. The latter response, however, was sensitive to a protein kinase A (PKA) inhibitor. Mutation of Gab1 Tyr627 to phenylalanine (YF-Gab1) to prevent the binding of SHP2 completely prevented the shear stress-induced phosphorylation of eNOS, leaving the Akt response intact. A dominant-negative SHP2 mutant prevented the activation of PKA and phosphorylation of eNOS without affecting that of Akt. Moreover, shear stress elicited the formation of a signalosome complex including eNOS, Gab1, SHP2 and the catalytic subunit of PKA. In isolated murine carotid arteries, flow-induced vasodilatation was prevented by a PKA inhibitor as well as by overexpression of either the YF-Gab1 or the dominant-negative SHP2 mutant. Thus, the shear stress-induced activation of eNOS depends on Gab1 and SHP2, which, in turn, regulate the phosphorylation and activity of eNOS by a PKA-dependent but Akt-independent mechanism.

Cyclic mechanical strain increases reactive oxygen species production in pulmonary epithelial cells.

Overdistention of lung tissue during mechanical ventilation may be one of the factors that initiates ventilator-induced lung injury (VILI). We hypothesized that cyclic mechanical stretch (CMS) of the lung epithelium is involved in the early events of VILI through the production of reactive oxygen species (ROS). Cultures of an immortalized human airway epithelial cell line (16HBE), a human alveolar type II cell line (A549), and primary cultures of rat alveolar type II cells were cyclically stretched, and the production of superoxide (O2-) was measured by dihydroethidium fluorescence. CMS stimulated increased production of O2- after 2 h in each type of cell. 16HBE cells exhibited no significant stimulation of ROS before 2 h of CMS (20% strain, 30 cycles/min), and ROS production returned to control levels after 24 h. Oxidation of glutathione (GSH), a cellular antioxidant, increased with CMS as measured by a decrease in the ratio of the reduced GSH level to the oxidized GSH level. Strain levels of 10% did not increase O2- production in 16HBE cells, whereas 15, 20, and 30% significantly increased generation of O2-. Rotenone, a mitochondrial complex I inhibitor, partially abrogated the stretch-induced generation of O2- after 2 h CMS in 16HBE cells. NADPH oxidase activity was increased after 2 h of CMS, contributing to the production of O2-. Increased ROS production in lung epithelial cells in response to elevated stretch may contribute to the onset of VILI.

Essential role of protein kinase G and decreased cytoplasmic Ca2+ levels in NO-induced inhibition of rat aortic smooth muscle cell motility.

Hyperinsulinemia is a major risk factor for the development of vascular disease. We have reported that insulin increases the motility of vascular smooth muscle cells via a hydrogen peroxide-mediated mechanism and that nitric oxide (NO) attenuates insulin-induced motility via a cGMP-mediated mechanism. Events downstream of cGMP elevation have not yet been investigated. The aim of our study was to test the hypothesis that antimotogenic effects of NO and cGMP in cultured rat aortic smooth muscle cells are mediated via PKG, followed by reduction of cytoplasmic Ca(2+) levels and increased protein tyrosine phosphatase-proline, glutamate, serine, and threonine activity, leading to suppression of agonist-induced elevation of hydrogen peroxide levels and cell motility. Treatment of primary cultures with adenovirus expressing PKG-1alpha mimicked NO-induced inhibition of insulin-elicited hydrogen peroxide elevation and cell motility, whereas treatment with the pharmacological PKG inhibitor Rp-8-bromo-3',5'-cyclic monophosphorothioate (Rp-8-Br-cGMPS) rescued the stimulatory effects of insulin that were suppressed by NO donor. Treatment of cells with insulin failed to increase cytoplasmic Ca(2+) levels, whereas NO donor decreased cytoplasmic Ca(2+) levels in the presence or absence of insulin. Treatment of cells with the Ca(2+) chelator BAPTA mimicked the effects of PKG and the NO donor and increased the activity of PTP-PEST. Finally, treatment with a dominant negative allele of PTP-PEST reversed the inhibitory effect of BAPTA on cell motility and hydrogen peroxide elevation. We conclude that NO-induced inhibition of cell motility occurs via PKG-mediated reduction of basal cytoplasmic Ca(2+) levels, followed by increased PTP-PEST activity, leading to decreased hydrogen peroxide levels and reduced cell motility.

Regulation of cell motility by tyrosine phosphorylated villin.

Temporal and spatial regulation of the actin cytoskeleton is vital for cell migration. Here, we show that an epithelial cell actin-binding protein, villin, plays a crucial role in this process. Overexpression of villin in doxycyline-regulated HeLa cells enhanced cell migration. Villin-induced cell migration was modestly augmented by growth factors. In contrast, tyrosine phosphorylation of villin and villin-induced cell migration was significantly inhibited by the src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) as well as by overexpression of a dominant negative mutant of c-src. These data suggest that phosphorylation of villin by c-src is involved in the actin cytoskeleton remodeling necessary for cell migration. We have previously shown that villin is tyrosine phosphorylated at four major sites. To further investigate the role of tyrosine phosphorylated villin in cell migration, we used phosphorylation site mutants (tyrosine to phenylalanine or tyrosine to glutamic acid) in HeLa cells. We determined that tyrosine phosphorylation at residues 60, 81, and 256 of human villin played an essential role in cell migration as well as in the reorganization of the actin cytoskeleton. Collectively, these studies define how biophysical events such as cell migration are actuated by biochemical signaling pathways involving tyrosine phosphorylation of actin binding proteins, in this case villin.

RhoA and Rac1 are both required for efficient wound closure of airway epithelial cells.

Repair of the airway epithelium after injury is critical for restoring normal lung. The reepithelialization process involves spreading and migration followed later by cell proliferation. Rho-GTPases are key components of the wound healing process in many different types of tissues, but the specific roles for RhoA and Rac1 vary and have not been identified in lung epithelial cells. We investigated whether RhoA and Rac1 regulate wound closure of bronchial epithelial cells. RhoA and Rac1 proteins were efficiently expressed in a cell line of human bronchial epithelial cells (16HBE) by adenovirus-based gene transfer. We found that both constitutively active RhoA and dominant negative RhoA inhibited wound healing, suggesting that both activation and inhibition of RhoA interfere with normal wound healing. Overexpression of wild-type Rac1 induced upregulation of RhoA, disrupted intercellular junctions, and inhibited wound closure. Dominant negative Rac1 also inhibited wound closure. Inhibition of the downstream effector of RhoA, Rho-kinase, with Y-27632 suppressed actin stress fibers and focal adhesion formation, increased Rac1 activity, and stimulated wound closure. The activity of both RhoA and Rac1 are influenced by the polymerization state of microtubules, and cell migration involves coordinated action of actin and microtubules. Microtubule depolymerization upon nocodazole treatment led to an increase in focal adhesions and decreased wound closure. We conclude that coordination of both RhoA and Rac1 activity contributes to bronchial epithelial wound repair mechanisms in vitro, that inhibition of Rho-kinase accelerates wound closure, and that efficient repair involves intact microtubules.