Anoxia-reoxygenation modulates cadmium-induced liver mitochondrial reactive oxygen species emission during oxidation of glycerol 3-phosphate.

Aquatic organisms are frequently exposed to multiple stressors including low dissolved oxygen (O2) and metals such as cadmium (Cd). Reduced O2 concentration and Cd exposure alter cellular function in part by impairing energy metabolism and dysregulating reactive oxygen species (ROS) homeostasis. However, little is known about the role of mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) in ROS homeostasis in fish and its response to environmental stress. In this study, mGPDH activity and the effects of anoxia-reoxygenation (A-RO) and Cd on ROS (as hydrogen peroxide, H2O2) emission in rainbow trout liver mitochondria during oxidation of glycerol 3-phosphate (G3P) were probed. Trout liver mitochondria exhibited low mGPDH activity that supported a low respiratory rate but substantial H2O2 emission rate. Cd evoked a low concentration stimulatory-high concentration inhibitory H2O2 emission pattern that was blunted by A-RO. At specific redox centers, Cd suppressed H2O2 emission from site IQ, but stimulated emission from sites IIIQo and GQ. In contrast, A-RO stimulated H2O2 emission from site IQ following 15 min exposure and augmented Cd-stimulated emission from site IIF after 30 min exposure but did not alter the rate of H2O2 emission from sites IIIQo and GQ. Additionally, Cd neither altered the activities of catalase, glutathione peroxidase, or thioredoxin reductase nor the concentrations of total glutathione, reduced glutathione, or oxidized glutathione. Overall, this study indicates that oxidation of G3P drives ROS production from mGPDH and complexes I, II and III, whereas Cd directly modulates redox sites but not antioxidant defense systems to alter mitochondrial H2O2 emission.

The intermediate-conductance calcium-activated potassium channel KCa3.1 contributes to alkalinization-induced vascular calcification in vitro.

OBJECTIVE: In order to find new strategies for the prevention of vascular calcification in uremic individuals especially treated by dialysis and develop novel therapeutic targets in vascular calcification, we explore the role of KCa3.1 in alkalinization-induced VSMCs calcification in vitro. METHOD: Rat VSMCs calcification model was established by beta-glycerophosphate (ß-GP, 10 mM) induction. The pH of Dulbecco's modified Eagle's medium (DMEM) was adjusted every 24 h with 10 mM HCl or 10 mM NaHCO3 . The mineralization was measured by Alizarin Red staining and O-cresolphthalein complex one method. mRNA and protein expression were detected by RT-PCR and Western blot or immunofluorescence. Ca2+ influx was measured by Elisa. RESULT: The results indicated that alkalization induced an increase in Ca2+ influx to enhance VSMCs calcification. Furthermore, the increase of calcification was associated with the expression of KCa3.1 via advanced expression of osteoblastic differentiation markers alkaline phosphatase (ALP) and Runt-related transcription factor 2 (Runx2). Blocking KCa3.1 with TRAM-34 or shRNA vector can significantly lowered the effects of calcification in the activity of ALP and Runx2 expression. CONCLUSION: Together all, our studies suggested that alkalinization can promote vascular calcification by upregulating KCa3.1 channel and enhancing osteogenic/chondrogenic differentiation by upregulating Runx2. The specific inhibitor TRAM-34 and KCa3.1-shRNA ameliorated VSMCs calcification by downregulating KCa3.1.

Label-free quantitative proteomics identifies Smarca4 is involved in vascular calcification.

Vascular calcification (VC) is a pathological process characterized by abnormal deposition of calcium phosphate, hydroxyapatite and other mineral substances in the vascular wall. Hyperphosphorus is an important risk factor associated with VC in the general population and patients with chronic kidney disease (CKD). However, there is still a lack of early biomarkers for hyperphosphorus induced VC. We established a calcific rat aorta vascular smooth muscle cells (RASMCs) model by stimulating with ß-glycerophosphate. Then we performed label-free quantitative proteomics combined with liquid chromatograph-mass spectrometer/mass spectrometer (LC-2D-MS/MS）analysis and bioinformatics analysis to find the potential biomarkers for VC. In the current study, we identified 113 significantly proteins. Fifty six of these proteins were significantly up-regulated and the other 57 proteins were significantly decreased in calcific RASMCs, compared to that of normal control cells (fold-change (fc)>1.2, p < .05). Bioinformatics analysis indicated that these significant proteins mainly involved in the placenta blood vessel development and liver regeneration. Their molecule function was cell adhesion molecule binding. Among them, Smarca4 is significantly up-regulated in calcific RASMCs with fc = 2.72 and p = .01. In addition, we also established VC rat model. Real-time quantitative PCR analysis confirmed that the expression of Smarca4 was significantly increased in the aorta of calcified rat. Consistent with the up-regulation of Smarca4, the expression of VC associated microRNA such as miR-133b and miR-155 was also increased. Consequently, our study demonstrates that Smarca4 is involved in hyperphosphorus-induced VC. This finding may contribute to the early diagnosis and prevention of VC.

An injectable and thermosensitive hydrogel: Promoting periodontal regeneration by controlled-release of aspirin and erythropoietin.

Periodontitis is an inflammatory disease induced by complex interactions between host immune system and plaque microorganism. Alveolar bone resorption caused by periodontitis is considered to be one of the main reasons for tooth loss in adults. To terminate the alveolar bone resorption, simultaneous anti-inflammation and periodontium regeneration is required, which has not appeared in the existing methods. In this study, chitosan (CS), ß-sodium glycerophosphate (ß-GP), and gelatin were used to prepare an injectable and thermosensitive hydrogel, which could continuously release aspirin and erythropoietin (EPO) to exert pharmacological effects of anti-inflammation and tissue regeneration, respectively. The releasing profile showed that aspirin and EPO could be continuously released from the hydrogels, which exhibited no toxicity both in vitro and in vivo, for at least 21 days. Immunohistochemistry staining and micro-CT analyses indicated that administration of CS/ß-GP/gelatin hydrogels loaded with aspirin/EPO could terminate the inflammation and recover the height of the alveolar bone, which is further confirmed by histological observations. Our results suggested that CS/ß-GP/gelatin hydrogels are easily prepared as drug-loading vectors with excellent biocompatibility, and the CS/ß-GP/gelatin hydrogels loaded with aspirin/EPO are quite effective in anti-inflammation and periodontium regeneration, which provides a great potential candidate for periodontitis treatment in the dental clinic. Statement of Significance To terminate the alveolar bone resorption caused by periodontitis, simultaneous anti-inflammation and periodontium regeneration is required, which has not appeared in the existing methods. Here, (1) the chitosan (CS)/ß-sodium glycerophosphate/gelatin hydrogels loaded with aspirin/erythropoietin (EPO) can form at body temperature in 5 min with excellent biocompatibility in vitro and in vivo; (2) The faster release of aspirin than EPO in the early stage is beneficial for anti-inflammation and provides a microenvironment for ensuring the regeneration function of EPO in the following step. In vivo experiments revealed that the hydrogels are effective in the control of inflammation and regeneration of the periodontium. These results indicate that our synthesized hydrogels have a great potential in the future clinical application.

Specific knockdown of WNT8b expression protects against phosphate-induced calcification in vascular smooth muscle cells by inhibiting the Wnt-ß-catenin signaling pathway.

In the last 10 years, the prevalence, significance, and regulatory mechanisms of vascular calcification (VC) have gained increasing recognition. The aim of this study is to explore the action of WNT8b in the development of phosphate-induced VC through its effect on vascular smooth muscle cells (VSMCs) in vitro by inactivating the Wnt-ß-catenin signaling pathway. To explore the effect of WNT8b on the Wnt-ß-catenin signaling pathway and VC in vitro, ß-glycerophosphate (GP)-induced T/G HA-VSMCs were treated with small interfering RNA against WNT8b (Si-WNT8b), Wnt-ß-catenin signaling pathway activator (LiCl) and both, respectively. Reverse transcription quantitative polymerase chain reaction and western blot analysis were used to determine the messenger RNA and protein levels of WNT8b, α-smooth muscle actin (α-SMA), calcification-associated molecules, and molecules related to the Wnt signaling pathway. The TOP/FOP-Flash reporter assay was performed to detect the transcription activity mediated by ß-catenin. Si-WNT8b reduced calcium deposition and the activity of alkaline phosphatase (ALP), increased the α-SMA level, and decreased bone morphogenetic protein 2, Pit1, MSX2, and Runt-related transcription factor 2 levels, whereas stimulation of LiCl worsened ß-GP-induced calcium deposition, increased the activity of ALP, and reduced the α-SMA expression level. Si-WNT8b reduced the levels of WNT8b, frizzled-4, ß-catenin, phospho-GSK-3ß (p-GSK-3ß), and cyclin-D, whereas it increased the levels of p-ß-catenin and GSK-3ß, indicating that si-WNT8b could alter the Wnt-ß-catenin signaling pathway and thus hamper the VC in T/G HA-VSMC, which was further demonstrated by the TOP/FOP-Flash assay and detection of the ß-catenin expression level in the nucleus. Altogether, we conclude that WNT8b knockdown terminates phosphate-induced VC in VSMCs by inhibiting the Wnt-ß-catenin signaling pathway.

The novel phospholipid mimetic KPC34 is highly active against preclinical models of Philadelphia chromosome positive acute lymphoblastic leukemia.

Philadelphia chromosome positive B cell acute lymphoblastic leukemia (Ph+ ALL) is an aggressive cancer of the bone marrow. The addition of tyrosine kinase inhibitors (TKIs) has improved outcomes but many patients still suffer relapse and novel therapeutic agents are needed. KPC34 is an orally available, novel phospholipid conjugate of gemcitabine, rationally designed to overcome multiple mechanisms of resistance, inhibit the classical and novel isoforms of protein kinase C, is able to cross the blood brain barrier and is orally bioavailable. KPC34 had an IC50 in the nanomolar range against multiple ALL cell lines tested but was lowest for Ph+ lines. In mice bearing either naïve or resistant Ph+ ALL, KPC34 treatment resulted in significantly improved survival compared to cytarabine and gemcitabine. Treatment with KPC34 and doxorubicin was more effective than doxorubicin and cytarabine. Mice with recurrence of their ALL after initial treatment with cytarabine and doxorubicin saw dramatic improvements in hind limb paralysis after treatment with KPC34 demonstrating activity against established CNS disease. Consistent with this KPC34 was better than gemcitabine at reducing CNS leukemic burden. These promising pre-clinical results justify the continued development of KPC34 for the treatment of Ph+ALL.

Hydrogen peroxide prevents vascular calcification induced ROS production by regulating Nrf-2 pathway.

BACKGROUND: Although vascular calcification in end-stage renal disease (ESRD) represents a ubiquitous human health problem, effective therapies with limited side effects are still lacking, and the precise mechanisms are not fully understood. The Nrf-2/ARE pathway is a pivotal to regulate anti-oxidative responses in vascular calcification upon ESRD. Although Nrf-2 plays a crucial role in atherosclerosis, pulmonary fibrosis, and brain ischemia, the effect of Nrf-2 and oxidative stress on vascular calcification in ESRD patients is still unclear. The aim of this research was to study the protective role of hydrogen peroxide in vascular calcification and the mechanism of Nrf-2 and oxidative stress on vascular calcification. MATERIALS AND METHODS: Here we used the rat vascular smooth muscle cell model of ß-glycerophosphate-induced calcification resembling vascular calcification in ESRD to investigate the therapeutic effect of 0.01 mM hydrogen peroxide on vascular calcification and further explores the possible underlying mechanisms. RESULTS: Our current report shows the in vitro role of 0.01 mM hydrogen peroxide in protecting against intracellular ROS accumulation upon vascular calcification. Both hydrogen peroxide and sulforaphane pretreatment reduced ROS production, increased the expression of Nrf-2, and decreased the expression of Runx2 following calcification. CONCLUSION: Our study demonstrates that 0.01 mM hydrogen peroxide can effectively protect rat aortic vascular smooth muscle cells against oxidative stress by preventing vascular calcification induced ROS production through Nrf-2 pathway. These data might define an antioxidant role of hydrogen peroxide in vascular calcification upon ESRD.

Preparation and characteristics of thermoresponsive gel of minocycline hydrochloride and evaluation of its effect on experimental periodontitis models.

In this study, a thermoresponsive gel for minocycline (MCL) with chitosan/ß-glycerophosphate (C/ß-GP) was formulated and its characterization, in vitro release, stability, toxicity and pharmacodynamics were investigated. The formulation containing MCL was prepared by pouring the chitosan solution directly onto the sterilized drug powder and stirring before mixing with the ß-glycerophosphate (ß-GP) solution. The final preparations contained 0.5% (w/v) chitosan, 1.8% (w/v) ß-GP and 2% (w/v) MCL. The drug content of prepared gels was in the range of 92-99%, and the pH value of the optimized formulation was found to be 5.6-6.2. The gelation temperature of the prepared C/ß-GP thermogelling solutions was 37 °C. Color, consistency, pH, viscosity and drug content of the in situ gels were found to be consistent, and no signs of separation and deterioration were observed over a period of 90 d. In vivo studies showed that rats' liver and kidney tissue sections were normal, with no structural damage. The constituents of the in situ gels formulation had a well-sustained release efficacy on the animal model of periodontitis.

A novel nontoxic inhibitor of the activation of NADPH oxidase reduces reactive oxygen species production in mouse lung.

1-Hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33) is a fluorinated phospholipid analog that inhibits the phospholipase A2 (PLA2) activity of peroxiredoxin 6 (Prdx6). Prdx6 PLA2 activity is required for activation of NADPH oxidase 2 and subsequent generation of reactive oxygen species (ROS). In vitro, MJ33 inhibited agonist-stimulated production of ROS by the isolated perfused mouse lung, lung microvascular endothelial cells, and polymorphonuclear leukocytes. MJ33 (0.02-0.5 µmol MJ33/kg body weight) in mixed unilamellar liposomes was administered to C57BL/6 mice by either intratracheal (i.t.) or i.v. routes. Lung MJ33 content, measured by liquid chromatography/mass spectroscopy, showed uptake of 67-87% of the injected dose for i.t. and 23-42% for i.v. administration at 4 hours postinjection. PLA2 activity of lung homogenates was markedly inhibited (>85%) at 4 hours postadministration. Both MJ33 content and PLA2 activity gradually returned to near control levels over the subsequent 24-72 hours. Mice treated with MJ33 at 12.5-25 µmol/kg did not show changes (compared with control) in clinical symptomatology, body weight, hematocrit, and histology of lung, liver, and kidney during a 30- to 50-day observation period. Thus, the toxic dose of MJ33 was >25 µmol/kg, whereas the PLA2 inhibitory dose was approximately 0.02 µmol/kg, indicating a high margin of safety. MJ33 administered to mice prior to lung isolation markedly reduced ROS production and tissue lipid and protein oxidation during ischemia followed by reperfusion. Thus, MJ33 could be useful as a therapeutic agent to prevent ROS-mediated tissue injury associated with lung inflammation or in harvested lungs prior to transplantation.

Thermogelling chitosan and collagen composite hydrogels initiated with beta-glycerophosphate for bone tissue engineering.

Chitosan and collagen type I are naturally derived materials used as cell carriers because of their ability to mimic the extracellular environment and direct cell function. In this study beta-glycerophosphate (beta-GP), an osteogenic medium supplement and a weak base, was used to simultaneously initiate gelation of pure chitosan, pure collagen, and chitosan-collagen composite materials at physiological pH and temperature. Adult human bone marrow-derived stem cells (hBMSC) encapsulated in such hydrogels at chitosan/collagen ratios of 100/0, 65/35, 25/75, and 0/100 wt% exhibited high viability at day 1 after encapsulation, but DNA content dropped by about half over 12 days in pure chitosan materials while it increased twofold in materials containing collagen. Collagen-containing materials compacted more strongly and were significantly stiffer than pure chitosan gels. In monolayer culture, exposure of hBMSC to beta-GP resulted in decreased cell metabolic activity that varied with concentration and exposure time, but washing effectively removed excess beta-GP from hydrogels. The presence of chitosan in materials resulted in higher expression of osterix and bone sialoprotein genes in medium with and without osteogenic supplements. Chitosan also increased alkaline phosphatase activity and calcium deposition in osteogenic medium. Chitosan-collagen composite materials have potential as matrices for cell encapsulation and delivery, or as in situ gel-forming materials for tissue repair.

[Structure of teichoic acid of a poly(glycerophosphate) nature from Streptomyces levoris K-3053 and its biological properties]. / Struktura teikhoevoi kisloty poli(glitserofosfatnoi) prirody Streptomyces levoris K-3053 i nekotorye ee biologicheskie svoistva.

The wall teichoic acid from S. levoris was studied. It is a poly(glycerophosphate)polymer consisting of 15-20 nonsubstituted glycerophosphate units coupled by the phosphodiether bonds according to the 1, 3 type. It was shown that the acid had antitumor activity and high cardiotoxicity evident from a clear-cut negative ionotropic effect on the cardiac papillar muscle in dogs.

Interaction of sn-glycerol 3-phosphorothioate with Escherichia coli: effect on cell growth and metabolism.

sn-Glycerol 3-phosphorothioate was found to be bacteriocidal to strains of Escherichia coli which have a functional sn-glycerol 3-phosphate transport system. This effect was manifest in strains 7 and 8, which are constitutive mutants for the utilization and transport of sn-glycerol 3-phosphate (glpRc2). Strain E15, which is considered to be wild type for the glycerol phosphate functional units, was affected by the phosphorothioate analog only under conditions that are known to induce the transport system for sn-glycerol 3-phosphate. In addition, another strain of E. coli, strain 6, which is isogenic with strain E15 but has an impaired sn-glycerol 3-phosphate transport system (glpT13), was not affected by similar concentrations of sn-glycerol 3-phosphorothioate. Transport studies in which [3H]glycerol phosphate and its phosphorothioate analog were used demonstrated that the latter compound was taken up via the specific active transport system for sn-glycerol 3-phosphate; the Km values were 9 and 11 microM, respectively. The rates of macromolecular synthesis were found to be inhibited severely by sn-glycerol 3-phosphorothioate at a concentration at which sn-glycerol 3-phosphate had no effect (5 microM). At a lower concentration of the analog (0.5 microM), the rates of protein synthesis and RNA synthesis (52 and 58% below control values after 90 min, respectively) were more sensitive than the rates of DNA synthesis and cell wall synthesis (18% below control values after 3 h for DNA; transient decrease in the cell wall values after 90 min). The levels of the nucleoside triphosphates were not affected by the presence of the phospholipid precursor or its analog at a concentration of 5 microM. The phospholipid composition was significantly altered in the presence of bacteriocidal concentrations (5 microM) of sn-glycerol 3-phosphorothioate. The amount of phosphatidylglycerol in the membranes decreased from 13.5 to 3.5%. Concomitant with this decrease in phosphatidylglycerol content was a fourfold increase in the 32P content of cardiolipin (from 6.8 to 24.2%), whereas the phosphatidylethanolamine content showed only a minor reduction (8%) after 3 h. The rates of synthesis of all of the phospholipids decreased in the presence of 5 microM sn-glycerol 3-phosphorothioate, with the most significant effects observed for phosphatidylglycerol (63% after 3 h). Phosphatidylglycerol showed increased rates of turnover after 90 min (21%) and 3 h (11%), with concomitant increases in the levels of cardiolipin of more than twofold. Our data suggest that a considerably greater proportion of phosphatidylglycerol turnover may be recover in cardiolipin than is metabolized via other pathways (e.g., the membrane-derived oligosaccharide pathway).

Effects of chronic beta-glycerophosphate administration on growth rate and serum, liver and bile lipid composition in the rat--a toxicity study.

The possible toxic effects of a high dose of orally administered disodium beta-glycerophosphate to the rat for a period of 33 weeks has been investigated. The studies revealed taht beta-glycerophosphate administration had no effect on either survival rat, body weight, hematological and liver function tests or on serum and liver lipids concentrations. All organ weights were similar in the control and experimental rats excepts for the kidneys which were significantly heavier in the beta-glycerophosphate-fed rats. The biliary phospholipids concentrations was significantly increased in the treated group as observed previously during short-term treatments. Histological examination of liver and kidneys did not reveal any pathological findings. These results suggest that long-term administration of beta-glycerophosphate did not induce any toxic manifestations. The observed hyperplasia of the kidneys was attributed to the effect of the sodium content of beta-glycerophosphate.