Endotoxin-tolerant rats are still protected from oxygen toxicity by low-dose endotoxin treatment

To determine if we could reduce endotoxin's potential for toxicity, we produced "endotoxin-tolerant" rats by administering progressively increasing daily doses of endotoxin (10 ng, 100 ng, 1 microgram, 10 micrograms/kg). This dosage regimen produced a high degree of tolerance to the toxic actions of endotoxin: whereas only 3/17 (18%) of control rats survived a normally lethal dose of endotoxin (25 mg/kg), survival for the endotoxin-tolerant rats was 16/16. When endotoxin-tolerant rats received a standard protective dose of 500 micrograms/kg endotoxin just before transfer to 96-98% O2, 19/20 survived the 72-h exposure period vs. 20-30% survival for controls. Thus whereas the endotoxin-tolerant state blocked the tested lethal and toxic effects of endotoxin, it did not nullify the O2 protective action of endotoxin. In addition, endotoxin's stimulatory effects on the lung antioxidant enzymes in the 96-98% O2-exposed rats was also not blocked by the endotoxin-tolerant state. Thus the therapeutic ratio (TR) of endotoxin as an experimental pharmacological treatment against O2-induced lung damage has been markedly enhanced (TR = ratio of dose producing beneficial effects to dose producing toxic effects).     

Quiescent fibroblasts exhibit high metabolic activity

Many cells in mammals exist in the state of quiescence, which is characterized by reversible exit from the cell cycle. Quiescent cells are widely reported to exhibit reduced size, nucleotide synthesis, and metabolic activity. Much lower glycolytic rates have been reported in quiescent compared with proliferating lymphocytes. In contrast, we show here that primary human fibroblasts continue to exhibit high metabolic rates when induced into quiescence via contact inhibition. By monitoring isotope labeling through metabolic pathways and quantitatively identifying fluxes from the data, we show that contact-inhibited fibroblasts utilize glucose in all branches of central carbon metabolism at rates similar to those of proliferating cells, with greater overflow flux from the pentose phosphate pathway back to glycolysis. Inhibition of the pentose phosphate pathway resulted in apoptosis preferentially in quiescent fibroblasts. By feeding the cells labeled glutamine, we also detected a "backwards" flux in the tricarboxylic acid cycle from α-ketoglutarate to citrate that was enhanced in contact-inhibited fibroblasts; this flux likely contributes to shuttling of NADPH from the mitochondrion to cytosol for redox defense or fatty acid synthesis. The high metabolic activity of the fibroblasts was directed in part toward breakdown and resynthesis of protein and lipid, and in part toward excretion of extracellular matrix proteins. Thus, reduced metabolic activity is not a hallmark of the quiescent state. Quiescent fibroblasts, relieved of the biosynthetic requirements associated with generating progeny, direct their metabolic activity to preservation of self integrity and alternative functions beneficial to the organism as a whole.     

Alpha-1B adrenergic receptor knockout mice are protected against methamphetamine toxicity

The psychostimulant methamphetamine (MA) is toxic to nigro-striatal dopaminergic terminals in both experimental animals and humans. In mice, three consecutive injections of MA (5 mg/kg, i.p. with 2 h of interval) induced a massive degeneration of the nigro-striatal pathway, as reflected by a 50% reduction in the striatal levels of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC), by a substantial reduction in striatal tyrosine hydroxylase and high-affinity DA transporter immunostaining, and by the development of reactive gliosis. MA-induced nigro-striatal degeneration was largely attenuated in mice lacking alpha1b-adrenergic receptors (ARs). MA-stimulated striatal DA release (measured by microdialysis in freely moving animals) and locomotor activity were also reduced in alpha1b-AR knockout mice. Pharmacological blockade of alpha-adrenergic receptors with prazosin also protected wild-type mice against MA toxicity. These results suggests that alpha1b-ARs may play a role in the toxicity of MA on nigro-striatal DA neurons.     

TRPV1-null mice are protected from diet-induced obesity

We explored a role for the capsaicin receptor, transient receptor potential channel vanilloid type 1 (TRPV1), in the regulation of feeding and body mass. On a 4.5% fat diet, wild-type and TRPV1-null mice gained equivalent body mass. On an 11% fat diet, however, TRPV1-null mice gained significantly less mass and adiposity; at 44 weeks the mean body weights of wild-type and TRPV1-null mice were approximately 51 and 34g, respectively. Both groups of mice consumed equivalent energy and absorbed similar amounts of lipids. TRPV1-null mice, however, exhibited a significantly greater thermogenic capacity. Interestingly, we found that 3T3-L1 preadipocytes expressed functional calcitonin gene-related peptide receptors. Thus, these data support a potential neurogenic mechanism by which TRPV1-sensitive sensory nerves may regulate energy and fat metabolism.     

Neonatal periostin knockout mice are protected from hyperoxia-induced alveolar simplication

In bronchopulmonary dysplasia (BPD), alveolar septae are thickened with collagen and α-smooth muscle actin, transforming growth factor (TGF)-β-positive myofibroblasts. Periostin, a secreted extracellular matrix protein, is involved in TGF-β-mediated fibrosis and myofibroblast differentiation. We hypothesized that periostin expression is required for hypoalveolarization and interstitial fibrosis in hyperoxia-exposed neonatal mice, an animal model for this disease. We also examined periostin expression in neonatal lung mesenchymal stromal cells and lung tissue of hyperoxia-exposed neonatal mice and human infants with BPD. Two-to-three day-old wild-type and periostin null mice were exposed to air or 75% oxygen for 14 days. Mesenchymal stromal cells were isolated from tracheal aspirates of premature infants. Hyperoxic exposure of neonatal mice increased alveolar wall periostin expression, particularly in areas of interstitial thickening. Periostin co-localized with α-smooth muscle actin, suggesting synthesis by myofibroblasts. A similar pattern was found in lung sections of infants dying of BPD. Unlike wild-type mice, hyperoxia-exposed periostin null mice did not show larger air spaces or α-smooth muscle-positive myofibroblasts. Compared to hyperoxia-exposed wild-type mice, hyperoxia-exposed periostin null mice also showed reduced lung mRNA expression of α-smooth muscle actin, elastin, CXCL1, CXCL2 and CCL4. TGF-β treatment increased mesenchymal stromal cell periostin expression, and periostin treatment increased TGF-β-mediated DNA synthesis and myofibroblast differentiation. We conclude that periostin expression is increased in the lungs of hyperoxia-exposed neonatal mice and infants with BPD, and is required for hyperoxia-induced hypoalveolarization and interstitial fibrosis.     

Autophagy induction rescues toxicity mediated by proteasome inhibition

The ubiquitin-proteasome and macroautophagy-lysosome pathways are major routes for intracytosolic protein degradation. In many systems, proteasome inhibition is toxic. A Nature article by Pandey et al. shows that this toxicity can be modulated by altering autophagic activity. Their tantalizing results suggest that overexpression of HDAC6 may increase flux through the autophagy pathway, thereby attenuating the toxicity resulting from proteasome inhibition.     

Cdc42-induced actin filaments are protected from capping protein.

Each actin filament has a pointed and a barbed end, however, filament elongation occurs primarily at the barbed end. Capping proteins, by binding to the barbed end, can terminate this elongation. The rate of capping depends on the concentration of capping protein [1], and thus, if capping terminates elongation, the length of filaments should vary inversely with the concentration of capping protein. In cell extracts, such as those derived from neutrophils, new actin filaments can be nucleated by addition of GTPgammaS-activated Cdc42 (a small GTPase of the Rho family). To determine whether elongation of these filaments is terminated by capping, we manipulated the concentration of capping protein, the major calcium-independent capping protein in neutrophils, and observed the effects on filament lengths. Depletion of 70% of the capping protein from extracts increased the mean length of filaments elongated from spectrin-actin seeds (very short actin filaments with free barbed ends) but did not increase the mean length of filaments induced by Cdc42. Furthermore, doubling the concentration of capping protein in cell extracts by adding pure capping protein did not decrease the mean length of filaments induced by Cdc42. These results suggest that the barbed ends of Cdc42-induced filaments are protected from capping by capping protein.     

Platelet-activating factor receptor knockout mice are protected from MPTP-induced dopaminergic degeneration

Platelet-activating factor (PAF), a potent mediator of inflammatory and immune responses, plays various roles in neuronal functions. However, little is known about the role of PAF/platelet-activating factor receptor (PAF-R) in Parkinson’s disease. Treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) resulted in significant increases in PAF species in the striatum of wild-type mice. These increases paralleled PAF-R gene expression in wild-type mice. Although nuclear factor kappa B (NF-κB) DNA-binding activity was increased significantly in MPTP-treated wild-type mice, this increase was not significant in PAF-R antagonist ginkgolide B (GB)-treated mice or PAF-R knockout (PAF-R^−/−) mice. Pyrrolidine dithiocarbamate (PDTC), an NF-κB inhibitor, significantly ameliorated the dopaminergic deficits induced by MPTP in wild-type mice. MPTP treatment significantly increased oxidative damage, the immunoreactivity of ionized calcium binding adaptor molecule 1 (Iba-1)-positive microglial cells, and microglial differentiation of the M1 type in the striatum of wild-type mice. Consistently, PDTC significantly attenuated MPTP-induced behavioral impairments in wild-type mice. However, dopaminergic deficits, oxidative damage, reactive microglial cells, and behavioral impairments induced by MPTP were not significantly observed in GB-treated mice or PAF-R^−/− mice. PDTC did not significantly alter the attenuations evident in MPTP-treated PAF-R^−/− mice, indicating that NF-κB is a critical target for neurotoxic modulation of PAF-R. We propose for the first time that PAF/PAF-R can mediate dopaminergic degeneration via an NF-κB-dependent signaling process.     

Synthetic liposomes are protective from bleomycin-induced lung toxicity

Idiopathic pulmonary fibrosis is a devastating disease characterized by a progressive, irreversible, and ultimately lethal form of lung fibrosis. Except for lung transplantation, no effective treatment options currently exist. The bleomycin animal model is one of the best studied models of lung injury and fibrosis. A previous study using mouse tumor models observed that liposome-encapsulated bleomycin exhibited reduced lung toxicity. Therefore, we hypothesized that airway delivery of synthetic phosphatidylcholine-containing liposomes alone would protect mice from bleomycin-induced lung toxicity. C57BL/6 mice were administered uncharged multilamellar liposomes (100 μl) or PBS vehicle on day 0 by airway delivery. Bleomycin (3.33 U/kg) or saline vehicle was then given intratracheally on day 1 followed by four additional separate doses of liposomes on days 4, 8, 12, and 16. Fluorescent images of liposomes labeled with 1,1'-dioctadecyl-3,3,3',3' tetramethylindocarbocyanine perchlorate confirmed effective and widespread delivery of liposomes to the lower respiratory tract as well as uptake primarily by alveolar macrophages and to a lesser extent by type II alveolar epithelial cells. Results at day 22, 3 wk after bleomycin treatment, showed that airway delivery of liposomes before and after intratracheal administration of bleomycin significantly reduced bleomycin-induced lung toxicity as evidenced by less body weight loss, chronic lung inflammation, and fibrosis as well as improved lung compliance compared with controls. These data indicate that airway-delivered synthetic liposomes represent a novel treatment strategy to reduce the lung toxicity associated with bleomycin in a mouse model.     

Platelet-activating factor receptor-deficient mice are protected from experimental sleep apnea-induced learning deficits

Intermittent hypoxia (IH) during sleep, a hallmark of sleep apnea, is associated with neurobehavioral impairments, regional neurodegeneration and increased oxidative stress and inflammation in rodents. Platelet-activating factor (PAF) is an important mediator of both normal neural plasticity and brain injury. We report that mice deficient in the cell surface receptor for PAF (PAFR-/-), a bioactive mediator of oxidative stress and inflammation, are protected from the spatial reference learning deficits associated with IH. Furthermore, PAFR-/- exhibit attenuated elevations in inflammatory signaling (cyclo-oxygenase-2 and inducible nitric oxide synthase activities), degradation of the ubiquitin-proteasome pathway and apoptosis observed in wild-type littermates (PAFR+/+) exposed to IH. Collectively, these findings indicate that inflammatory signaling and neurobehavioral impairments induced by IH are mediated through PAF receptors.     

Mice lacking functional TRPV1 are protected from pressure overload cardiac hypertrophy

TRPV1 (transient receptor potential cation channel, subfamily V, member 1) is best studied in peripheral sensory neurons as a pain receptor; however TRPV1 is expressed in numerous tissues and cell types including those of the cardiovascular system. TRPV1 expression is upregulated in the hypertrophic heart, and the channel is positioned to receive stimulatory signals in the hypertrophic heart. We hypothesized that TRPV1 has a role in regulating cardiac hypertrophy. Using transverse aortic constriction to model pressure overload cardiac hypertrophy we show that mice lacking functional TRPV1, compared to wild type, have improved heart function, and reduced hypertrophic, fibrotic and apoptotic markers. This suggests that TRPV1 plays a role in the progression of cardiac hypertrophy, and presents a possible therapeutic target for the treatment of cardiac hypertrophy and heart failure.     

Nmur1 mice are not protected from cutaneous inflammation

Neuromedin U (Nmu) is a neuropeptide expressed primarily in the gastrointestinal tract and central nervous system. Previous reports have identified two G protein-coupled receptors (designated Nmur1 and Nmur2) that bind Nmu. Recent reports suggest that Nmu mediates immune responses involving mast cells, and Nmur1 has been proposed to mediate these responses. In this study, we generated mice with an Nmur1 deletion and then profiled the responses of these mice in a cutaneous inflammation model utilizing complete Freund’s adjuvant (CFA). We report here that mice lacking Nmur1 had normal inflammation responses with moderate changes in serum cytokines compared to Nmur1^+/+ littermates. Although differences in IL-6 were observed in mice lacking Nmu peptide, these mice exhibited a normal response to CFA. Our data argues against a major role for Nmur1 in mediating the reported inflammatory functions of NmU.     

Plasminogen activator inhibitor-1 deficient mice are protected from angiotensin II-induced fibrosis

PAI-1 has been shown to be both profibrotic and antifibrotic in animal models of hepatic fibrosis. Although these models have similarities to human fibrotic liver disease, no rodent model completely recapitulates the clinical situation; indeed, transaminase values in most models of hepatic fibrosis are much higher than in chronic liver diseases in humans. Here, wild-type and PAI-1^−/− mice were administered AngII (500 ng/kg/min) for 4 weeks. ECM accumulation was evaluated by Sirius red staining, hydroxyproline content, and fibrin and collagen immunostaining. Induction of pro-fibrotic genes was assessed by real-time RT-PCR. Despite the absence of any significant liver damage, AngII infusion increased the deposition of hepatic collagen and fibrin ECM, with a perisinusoidal pattern. PAI-1^−/− mice were protected from these ECM changes, indicating a causal role of PAI-1 in this fibrosis model. Protection in the knockout strain correlated with a blunted increase in αSMA, and elevated activities of matrix metalloproteinases (MMP2, MMP9). These data suggest that PAI-1 plays a critical role in mediating fibrosis caused by AngII and lends weight-of-evidence to a pro-fibrotic role of this protein in liver. Furthermore, the current study proposes a new model of ‘pure’ hepatic fibrosis in mice with little inflammation or hepatocyte death.     

Plasminogen activator inhibitor-1 deficient mice are protected from angiotensin II-induced fibrosis

Fungal methionine synthase, Met6p, transfers a methyl group from 5-methyl-tetrahydrofolate to homocysteine to generate methionine. The enzyme is essential to fungal growth and is a potential anti-fungal drug design target. We have characterized the enzyme from the pathogen Candida albicans but were unable to crystallize it in native form. We converted Lys103, Lys104, and Glu107 all to Tyr (Met6pY), Thr (Met6pT) and Ala (Met6pA). All variants showed wild-type kinetic activity and formed useful crystals, each with unique crystal packing. In each case the mutated residues participated in beneficial crystal contacts. We have solved the three structures at 2.0–2.8 Å resolution and analyzed crystal packing, active-site residues, and similarity to other known methionine synthase structures. C. albicans Met6p has a two domain structure with each of the domains having a (βα)₈-barrel fold. The barrels are arranged face-to-face and the active site is located in a cleft between the two domains. Met6p utilizes a zinc ion for catalysis that is bound in the C-terminal domain and ligated by four conserved residues: His657, Cys659, Glu679 and Cys739.     

Mice lacking functional TRPV1 are protected from pressure overload cardiac hypertrophy

TRPV1 (transient receptor potential cation channel, subfamily V, member 1) is best studied in peripheral sensory neurons as a pain receptor; however TRPV1 is expressed in numerous tissues and cell types including those of the cardiovascular system. TRPV1 expression is upregulated in the hypertrophic heart, and the channel is positioned to receive stimulatory signals in the hypertrophic heart. We hypothesized that TRPV1 has a role in regulating cardiac hypertrophy. Using transverse aortic constriction to model pressure overload cardiac hypertrophy we show that mice lacking functional TRPV1, compared to wild type, have improved heart function, and reduced hypertrophic, fibrotic and apoptotic markers. This suggests that TRPV1 plays a role in the progression of cardiac hypertrophy, and presents a possible therapeutic target for the treatment of cardiac hypertrophy and heart failure.     

Cyclic nucleotide-gated ion channels in rod photoreceptors are protected from retinoid inhibition

In vertebrate rods, photoisomerization of the 11-cis retinal chromophore of rhodopsin to the all-trans conformation initiates a biochemical cascade that closes cGMP-gated channels and hyperpolarizes the cell. All-trans retinal is reduced to retinol and then removed to the pigment epithelium. The pigment epithelium supplies fresh 11-cis retinal to regenerate rhodopsin. The recent discovery that tens of nanomolar retinal inhibits cloned cGMP-gated channels at low [cGMP] raised the question of whether retinoid traffic across the plasma membrane of the rod might participate in the signaling of light. Native channels in excised patches from rods were very sensitive to retinoid inhibition. Perfusion of intact rods with exogenous 9- or 11-cis retinal closed cGMP-gated channels but required higher than expected concentrations. Channels reopened after perfusing the rod with cellular retinoid binding protein II. PDE activity, flash response kinetics, and relative sensitivity were unchanged, ruling out pharmacological activation of the phototransduction cascade. Bleaching of rhodopsin to create all-trans retinal and retinol inside the rod did not produce any measurable channel inhibition. Exposure of a bleached rod to 9- or 11-cis retinal did not elicit channel inhibition during the period of rhodopsin regeneration. Microspectrophotometric measurements showed that exogenous 9- or 11-cis retinal rapidly cross the plasma membrane of bleached rods and regenerate their rhodopsin. Although dark-adapted rods could also take up large quantities of 9-cis retinal, which they converted to retinol, the time course was slow. Apparently cGMP-gated channels in intact rods are protected from the inhibitory effects of retinoids that cross the plasma membrane by a large-capacity buffer. Opsin, with its chromophore binding pocket occupied (rhodopsin) or vacant, may be an important component. Exceptionally high retinoid levels, e.g., associated with some retinal degenerations, could overcome the buffer, however, and impair sensitivity or delay the recovery after exposure to bright light.     

L-Glutamate toxicity in Huntington's disease fibroblasts

Brain degeneration in Huntington's Disease is thought to occur primarily in the regions of high L-glutamate concentrations. Huntington's Disease fibroblast cultures have a sensitivity to these high concentrations of L-glutamate. These cells show degeneration and loss of viability, within 12 hrs, following treatment with 30 mM L-glutamate. This effect appears to be specific for L-glutamate, can be prevented by glutamine, and is not observed in matched control cultures. The observed glutamate sensitivity may serve as a biochemical genetic marker and the excessive effect of glutamate on neuronal membranes could initiate the characteristic neuronal degeneration observed in Huntington's Disease brains.     

Lovastatin and simvastatin are modulators of the proteasome

Lovastatin and simvastatin are HMG-CoA reductase inhibitors widely used as antihyperlipidemic drugs, which also display antiproliferative properties. In the present paper, we provide evidence that both lovastatin and simvastatin are modulators of the purified bovine pituitary 20 S proteasome, since they mildly stimulate the chymotrypsin-like activity and inhibit the peptidylglutamylpeptide hydrolyzing activity without interfering with the trypsin-like activity. However, those effects are only observed when the closed ring forms of the drugs are used, while the opened ring form of lovastatin acts as a mild inhibitor of the chymotrypsin like activity. The closed ring form of lovastatin is much more potent as a cytotoxic agent on the Colon-26 (C-26) colon carcinoma cell line than the opened ring form, which is only mildly cytostatic. Moreover, neither the cytotoxic effects nor the effects on 20 S proteasome activities are prevented by mevalonate, which by itself inhibits the trypsin-like activity of the proteasome. Neither the opened ring nor the closed ring form of lovastatin induces an accumulation of ubiquitin-protein conjugates, which is observed after treatment with lactacystin, a selective proteasome inhibitor. In contrast with the opened ring form of lovastatin, the closed ring form induces the disappearance of detectable p27(kip1) from C-26 cells. Altogether, our results indicate that the closed ring form of lovastatin induces cytotoxic effects independent of its HMG-CoA inhibiting activity, however, those effects are mediated by a complex modulation of proteasome activity rather than by inhibition of the 20 S proteasome.