d-609 and Disease-Models--Animal

Accumulated oxidative damage may lead to irreversible retinal pigmented epithelium (RPE) cell death, which is considered to be the primary cause of dry age-related macular degeneration (AMD), leading to blindness in the elderly. However, an effective therapy for this disease is lacking. Here, we described a robust high-content screening procedure with a library of 814 protective compounds and found that D609 strongly protected RPE cells from sodium iodate (SI)-induced oxidative cell death and prolonged their healthy survival. D609 effectively attenuated excessive reactive oxygen species (ROS) and prevented severe mitochondrial loss due to oxidative stress in the RPE cells. Surprisingly, the potent antioxidative effects of D609 were not achieved through its own reducibility but were primarily dependent on its ability to increase the expression of metallothionein. The injection of this small water-soluble molecule also showed an explicit protective effect of the RPE layer in an SI-induced AMD mouse model. These findings suggested that D609 could serve as a novel antioxidative protector of RPE cells both in vitro and in vivo and unveiled a novel antioxidative mechanism of D609, which may ultimately have clinical applications for the treatment of AMD.

Topics: Animals; Cell Death; Disease Models, Animal; Humans; Macular Degeneration; Mitochondria; Norbornanes; Oxidative Stress; Reactive Oxygen Species; Retinal Pigment Epithelium; Thiocarbamates

Pulmonary hypertension (PH) is frequently observed in patients with acute respiratory distress syndrome (ARDS) and it is associated with an increased risk of mortality. Both acid sphingomyelinase (aSMase) activity and interleukin 6 (IL-6) levels are increased in patients with sepsis and correlate with worst outcomes, but their role in pulmonary vascular dysfunction pathogenesis has not yet been elucidated. Therefore, the aim of this study was to determine the potential contribution of aSMase and IL-6 in the pulmonary vascular dysfunction induced by lipopolysaccharide (LPS).. Rat or human pulmonary arteries (PAs) or their cultured smooth muscle cells (SMCs) were exposed to LPS, SMase or IL-6 in the absence or presence of a range of pharmacological inhibitors. The effects of aSMase inhibition in vivo with D609 on pulmonary arterial pressure and inflammation were assessed following intratracheal administration of LPS.. LPS increased ceramide and IL-6 production in rat pulmonary artery smooth muscle cells (PASMCs) and inhibited pulmonary vasoconstriction induced by phenylephrine or hypoxia (HPV), induced endothelial dysfunction and potentiated the contractile responses to serotonin. Exogenous SMase and IL-6 mimicked the effects of LPS on endothelial dysfunction, HPV failure and hyperresponsiveness to serotonin in PA; whereas blockade of aSMase or IL-6 prevented LPS-induced effects. Finally, administration of the aSMase inhibitor D609 limited the development of endotoxin-induced PH and ventilation-perfusion mismatch. The protective effects of D609 were validated in isolated human PAs.. Our data indicate that aSMase and IL-6 are not simply biomarkers of poor outcomes but pathogenic mediators of pulmonary vascular dysfunction in ARDS secondary to Gram-negative infections.

Topics: Animals; Bridged-Ring Compounds; Cells, Cultured; Ceramidases; Disease Models, Animal; Fluorescent Antibody Technique; Humans; Hypertension, Pulmonary; Interleukin-1beta; Interleukin-6; Lipopolysaccharides; Male; Muscle, Smooth, Vascular; Norbornanes; Pulmonary Artery; Rats; Rats, Wistar; Sphingomyelin Phosphodiesterase; Thiocarbamates; Thiones; Vasoconstriction

Neuron apoptosis and inflammatory responses contribute to subarachnoid hemorrhage (SAH)-induced early brain injury (EBI), which is the main aspect that affects patients' outcome. Previous research has demonstrated that phosphatidylcholine-specific phospholipase C (PC-PLC) plays critical roles in cell apoptosis and various inflammatory responses, and that tricyclodecan-9-yl-xanthogenate (D609), a well known PC-PLC inhibitor, is a powerful agent to protect brain from cerebral ischemic injury and SAH-induced cerebral vasospasm. However, the association between PC-PLC and SAH-induced EBI is undetermined. Therefore, we sought to investigate whether PC-PLC was implicated in SAH-induced EBI. Compared with sham group, an upregulation of PC-PLC activity was detected in the brain tissue and serum of SAH group. Pharmacological blockade of PC-PLC by D609 attenuated neurological behavior impairment, brain edema and blood-brain barrier (BBB) damage induced by SAH. In addition, D609 treatment significantly inhibited SAH-induced inflammatory response and neuron apoptosis. Furthermore, inhibition of PC-PLC in primary-cultured rat cortical neurons attenuated oxyhemoglobin (OxyHb)-induced apoptosis morphology and decrease in survival rate. In conclusion, our data suggest that PC-PLC participates in SAH-induced EBI.

Topics: Animals; Antioxidants; Blood-Brain Barrier; Brain; Brain Edema; Bridged-Ring Compounds; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; Gene Expression Regulation, Enzymologic; Male; Nervous System Diseases; Neurons; Norbornanes; Oxyhemoglobins; Phosphopyruvate Hydratase; Rats; Rats, Sprague-Dawley; Subarachnoid Hemorrhage; Thiocarbamates; Thiones; Time Factors; Type C Phospholipases; von Willebrand Factor

Atherosclerosis is considered to be a chronic inflammatory disease. Previous research has demonstrated that phosphatidylcholine-specific phospholipase C (PC-PLC) plays critical roles in various inflammatory responses. However, the association between PC-PLC and atherosclerosis is undetermined. Therefore, we sought to investigate whether PC-PLC was implicated in atherosclerosis.. Immunofluorescence analysis revealed an upregulation of PC-PLC in the aortic endothelium from apolipoprotein E-deficient (apoE(-/-)) mice. PC-PLC level and activity were also increased in human umbilical vein endothelial cells in response to oxidized low-density lipoprotein treatment. Pharmacological blockade of PC-PLC by D609 inhibited the progression of preexisting atherosclerotic lesions in apoE(-/-) mice and changed the lesion composition into a more stable phenotype. Using a combination of pharmacological inhibition, polyclonal antibodies, confocal laser scanning microscopy and Western blotting, we demonstrated that PC-PLC was required for endothelial expression of lectin-like oxidized low-density lipoprotein receptor-1. In addition, D609 treatment significantly decreased the aortic endothelial expression of the vascular cell adhesion molecule-1 and the intercellular adhesion molecule-1. Furthermore, inhibition of PC-PLC in human umbilical vein endothelial cells reduced the oxidized low-density lipoprotein induced expression of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and monocyte chemotactic protein-1.. Our data suggest that PC-PLC contributes to the progression of atherosclerosis.

Topics: Animals; Apolipoproteins E; Atherosclerosis; Bridged-Ring Compounds; Cells, Cultured; Chemokine CCL2; Disease Models, Animal; Disease Progression; Endothelium, Vascular; Humans; Intercellular Adhesion Molecule-1; Lipoproteins, LDL; Mice; Mice, Knockout; Norbornanes; Thiocarbamates; Thiones; Type C Phospholipases; Up-Regulation; Vascular Cell Adhesion Molecule-1

To evaluate possible effects of the intracerebroventricular (icv) injection of either O-Tricyclo [5.2.1.0(2,6)] dec-9-yl dithiocarbonate potassium salt (D609), a potent antioxidant and inhibitor of phosphatidylcholine specific phospholipase C (PtdCho-PLC) and acid sphingomyelinase (ASMase), or the spin trap/free radical scavenger N-tert-Butyl-alpha-phenylnitrone (PBN), on mechanical allodynia induced by facial carrageenan injection in mice.. Balb/c mice received icy injection of D609/PBN plus facial carrageenan injection, and the number of face wash strokes to von Frey hair mechanical stimulation of the maxillary skin was quantified. PtdCho-PLC and ASMase activities were also assayed in the brainstem, thalamus, and somatosensory cortex.. Mice that received the icy injection of 10 nmol D609 plus facial carrageenan injection showed significantly fewer face wash strokes evoked by von Frey hair stimulation (indicating reduced mechanical allodynia) at 1 and 3 days post-injection, compared to mice that received icy injection of isotonic saline plus facial carrageenan injection. Mice that received icy injection of 1.13 micromol PBN plus facial carrageenan injection likewise showed significantly fewer face wash strokes after facial carrageenan injection, compared to isotonic saline-injected plus carrageenan-injected controls. D609 injection also resulted in significantly reduced ASMase activity in the brainstem, thalamus, and somatosensory cortex 3 days after injection, compared to controls.. The icv injections of D609 and PBN were effective in reducing mechanical allodynia after facial carrageenan injection-induced pain. Together, the results point to a possible role of central nervous system sphingolipids and/or free radicals in orofacial pain.

Topics: Animals; Antioxidants; Brain; Brain Stem; Bridged-Ring Compounds; Carrageenan; Cerebral Ventricles; Cyclic N-Oxides; Disease Models, Animal; Facial Pain; Free Radical Scavengers; Injections, Intraventricular; Male; Maxillary Nerve; Mice; Mice, Inbred BALB C; Norbornanes; Somatosensory Cortex; Sphingolipids; Sphingomyelin Phosphodiesterase; Stimulation, Chemical; Thalamus; Thiocarbamates; Thiones; Trigeminal Ganglion; Type C Phospholipases

Despite 2-methoxyestradiol (2ME2) and tricyclodecan-9-yl-xanthogenate (D609) having multiple effects on cancer cells, mechanistically, both of them down-regulate hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF). We hypothesize HIF-1alpha plays an essential role in cerebral ischemia as a pro-apoptosis regulator; 2ME2 and D609 decrease the levels of HIF-1alpha and VEGF, that might contribute to protecting brain from ischemia injury. A total of 102 male Sprague-Dawley rats were split into five groups: sham, middle cerebral artery occlusion (MCAO), MCAO + dimethyl sulfoxide, MCAO + 2ME2, and MCAO + D609. 2ME2 and D609 were injected intraperitoneally 1 h after reperfusion. Rats were killed at 24 h and 7 days. At 24 h, 2ME2 and D609 reduce the levels of HIF-1alpha and VEGF (enzyme-linked immunosorbent assay), depress the expression of HIF-1alpha, VEGF, BCL2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) and cleaved caspase 3 (western blot and immunohistochemistry) in the brain infarct area. Double fluorescence labeling shows HIF-1alpha positive immunoreactive materials are co-localized with BNIP3 and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling inside the nuclei of neurons. At 7 days, 2ME2 and D609 reduce the infarct volume (2,3,7-triphenyltetrazolium chloride) and blood-brain barrier extravasation, decrease the mortality and improve the neurological deficits. In conclusion, 2ME2 and D609 are powerful agents to protect brain from cerebral ischemic injury by inhibiting HIF-1alpha expression, attenuating the superfluous expression of VEGF to avoid blood-brain barrier disruption and suppressing neuronal apoptosis via BNIP3 pathway.

Topics: 2-Methoxyestradiol; Animals; Apoptosis; Blood-Brain Barrier; Brain Edema; Brain Infarction; Brain Ischemia; Bridged-Ring Compounds; Caspase 3; Cerebral Cortex; Disease Models, Animal; Down-Regulation; Estradiol; Hypoxia-Inducible Factor 1, alpha Subunit; In Situ Nick-End Labeling; Infarction, Middle Cerebral Artery; Male; Membrane Proteins; Mitochondrial Proteins; Neuroprotective Agents; Norbornanes; Proto-Oncogene Proteins; Rats; Rats, Sprague-Dawley; Signal Transduction; Thiocarbamates; Thiones; Treatment Outcome; Vascular Endothelial Growth Factor A

Considerable evidence supports the role of oxidative stress in the pathogenesis of Alzheimer's disease. One hallmark of Alzheimer's disease is the accumulation of amyloid beta-peptide, which invokes a cascade of oxidative damage to neurons that can eventually result in neuronal death. Amyloid beta-peptide is the main component of senile plaques and generates free radicals ultimately leading to neuronal damage of membrane lipids, proteins and nucleic acids. Therefore, interest in the protective role of different antioxidant compounds has been growing for treatment of Alzheimer's disease and other oxidative stress-related disorders. Among different antioxidant drugs, much interest has been devoted to "thiol-delivering" compounds. Tricyclodecan-9-yl-xanthogenate is an inhibitor of phosphatidylcholine specific phospholipase C, and recent studies reported its ability to act as a glutathione-mimetic compound. In the present study, we investigate the in vivo ability of tricyclodecan-9-yl-xanthogenate to protect synaptosomes against amyloid beta-peptide-induced oxidative stress. Gerbils were injected i.p. with tricyclodecan-9-yl-xanthogenate or with saline solution, and synaptosomes were isolated from the brain. Synaptosomal preparations isolated from tricyclodecan-9-yl-xanthogenate injected gerbils and treated ex vivo with amyloid beta-peptide (1-42) showed a significant decrease of oxidative stress parameters: reactive oxygen species levels, protein oxidation (protein carbonyl and 3-nitrotyrosine levels) and lipid peroxidation (4-hydroxy-2-nonenal levels). Our results are consistent with the hypothesis that modulation of free radicals generated by amyloid beta-peptide might represent an efficient therapeutic strategy for treatment of Alzheimer's disease and other oxidative-stress related disorders. Based on the above data, we suggest that tricyclodecan-9-yl-xanthogenate is a potent antioxidant and could be of importance for the treatment of Alzheimer's disease and other oxidative stress-related disorders.

Topics: Aldehydes; Alzheimer Disease; Amyloid beta-Peptides; Animals; Antioxidants; Brain; Bridged-Ring Compounds; Disease Models, Animal; Free Radicals; Gerbillinae; Lipid Peroxidation; Male; Nerve Degeneration; Neurons; Norbornanes; Oxidative Stress; Peptide Fragments; Reactive Oxygen Species; Synaptosomes; Thiocarbamates; Thiones; Type C Phospholipases; Tyrosine

We investigated the alteration of signal transduction after acute esophagitis in cat lower esophageal sphincter (LES). Acute esophagitis (AE) was induced by perfusion with 0.1N HCl at a rate of 1 ml/min for 45 min over three consecutive days. Acetylcholine (ACh)-induced contraction was inhibited by M3>> M1 or M2 antagonists in normal LES. In AE, inhibition by M2 antagonists increased significantly, so that contraction was inhibited by M3> M2> M1 antagonists and the expression of M2 and M3 receptors were increased when compared to normal LES. In normal cells, ACh-induced contractions were antagonized by antibody against G(q/11) and the phosphatidylinositol-specific phospholipase C (PI-PLC) antagonist, U73122. The phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor, D609, or the phospholipase D inhibitor, propranolol had no effects on contraction in normal LES. However, in AE, G(q/11), and G(i3) antibodies reduced ACh-induced contraction and U73122, propranolol and D609 also reduced the contraction. In AE, we found that the expressions of G protein subtypes were increased but the expression of PLCbeta1, and PLCgamma1 were decreased when compared to normal LES. In conclusion, experimental esophagitis may alter the signal transduction by ACh in LES. ACh-induced contraction is mediated by M3 receptor, G(q/11) and PI-PLC in normal LES. However, in AE, the contractions are mediated by M2, M3 receptor, G(q/11) and G(i3). PC-PLC and PLD as PI-PLC are also involved in ACh-induced cell contraction in AE.

Topics: Acetylcholine; Acute Disease; Animals; Bridged-Ring Compounds; Cats; Disease Models, Animal; Drug Antagonism; Esophagitis; Esophagogastric Junction; Estrenes; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Norbornanes; Phosphatidylinositol Diacylglycerol-Lyase; Phosphodiesterase Inhibitors; Phosphoinositide Phospholipase C; Propranolol; Pyrrolidinones; Receptors, Muscarinic; Signal Transduction; Thiocarbamates; Thiones

Stroke is a major cause of long-term disability, the severity of which is directly related to the numbers of neurons that succumb to the ischemic insult. The signaling cascades activated by cerebral ischemia that may either promote or protect against neuronal death are not well understood. One injury-responsive signaling pathway that has recently been characterized in studies of non-neural cells involves cleavage of membrane sphingomyelin by acidic and/or neutral sphingomyelinase (ASMase) resulting in generation of the second messenger ceramide. We now report that transient focal cerebral ischemia induces large increases in ASMase activity, ceramide levels, and production of inflammatory cytokines in wild-type mice, but not in mice lacking ASMase. The extent of brain tissue damage is decreased and behavioral outcome improved in mice lacking ASMase. Neurons lacking ASMase exhibit decreased vulnerability to excitotoxicity and hypoxia, which is associated with decreased levels of intracellular calcium and oxyradicals. Treatment of mice with a drug that inhibits ASMase activity and ceramide production reduces ischemic neuronal injury and improves behavioral outcome, suggesting that drugs that inhibit this signaling pathway may prove beneficial in stroke patients.

Topics: Animals; Apoptosis; Brain; Bridged-Ring Compounds; Calcium; Cell Survival; Ceramides; Cerebral Cortex; Cerebral Infarction; Cytokines; Disease Models, Animal; Enzyme Activation; Homeostasis; Ischemic Attack, Transient; Kinetics; Mice; Mice, Knockout; Neurons; Norbornanes; Phosphodiesterase Inhibitors; Second Messenger Systems; Sphingomyelin Phosphodiesterase; Thiocarbamates; Thiones