Publications by authors named "James A Thliveris"

21 Publications

  • Page 1 of 1

Impact of Wnt/β-catenin signaling on ethanol-induced changes in brain endothelial cell permeability.

J Neurochem 2020 Sep 30. Epub 2020 Sep 30.

Department of Pharmacology and Therapeutics, Max Rady College of Medicine, University of Manitoba, Winnipeg, Canada.

Chronic exposure to ethanol is associated with enhanced leakiness in the brain microvessel endothelial cells that form the blood-brain barrier (BBB). As previous studies suggested Wnt/β-catenin signaling could improve the BBB phenotype of brain endothelial cells, we examined the extent to which Wnt signaling is altered following ethanol exposure, using both a cell culture model of the BBB and mice exposed to ethanol, and the ability of Wnt activation to reverse the permeability effects of ethanol. The human brain endothelial cells, hCMEC/D3, were exposed to ethanol (17-200 mM) for various periods of time (0-96 hr) and Wnt signaling, as well as expression of downstream genes influencing BBB integrity in the cell monolayers were monitored. Determination of Wnt signaling in both brain homogenates and brain microvessels from mice exposed to ethanol was also performed. The effects of ethanol on the permeability of the hCMEC/D3 monolayers were examined using both small molecular weight (sodium fluorescein) and large molecular weight (IRdye 800CW PEG) fluorescent markers. Exposure of hCMEC/D3 to ethanol (50 mM) caused a down-regulation of Wnt/β-catenin signaling, a reduction of tight junction protein expression and up-regulation of plasmalemma vesicle associated protein (PLVAP). A similar reduction in Wnt/β-catenin activity in both cortical brain homogenates and isolated cortical cerebral microvessels were observed in mice. Other areas such as cerebellum and striatum displayed as much as 3-6 fold increases in Dkk-1, an endogenous Wnt inhibitor. Ethanol exposure caused significant changes in both sodium fluorescein and IRdye 800CW PEG permeability (2-fold compared to control). The ethanol-induced increases in permeability were attenuated by treatment with known Wnt activators (i.e. LiCl or Wnt3a). Additional screens of CNS active agents with possible Wnt activity indicated fluoxetine could also prevent the permeability effects of ethanol. These studies suggest that ethanol-induced changes in brain microvessel permeability can be reversed through activation of Wnt signaling.
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http://dx.doi.org/10.1111/jnc.15203DOI Listing
September 2020

Doxorubicin-loaded iron oxide nanoparticles for glioblastoma therapy: a combinational approach for enhanced delivery of nanoparticles.

Sci Rep 2020 07 9;10(1):11292. Epub 2020 Jul 9.

Department of Biomedical Engineering, University of Manitoba, Winnipeg, MB, Canada.

Although doxorubicin (DOX) is an effective anti-cancer drug with cytotoxicity in a variety of different tumors, its effectiveness in treating glioblastoma multiforme (GBM) is constrained by insufficient penetration across the blood-brain barrier (BBB). In this study, biocompatible magnetic iron oxide nanoparticles (IONPs) stabilized with trimethoxysilylpropyl-ethylenediamine triacetic acid (EDT) were developed as a carrier of DOX for GBM chemotherapy. The DOX-loaded EDT-IONPs (DOX-EDT-IONPs) released DOX within 4 days with the capability of an accelerated release in acidic microenvironments. The DOX-loaded EDT-IONPs (DOX-EDT-IONPs) demonstrated an efficient uptake in mouse brain-derived microvessel endothelial, bEnd.3, Madin-Darby canine kidney transfected with multi-drug resistant protein 1 (MDCK-MDR1), and human U251 GBM cells. The DOX-EDT-IONPs could augment DOX's uptake in U251 cells by 2.8-fold and significantly inhibited U251 cell proliferation. Moreover, the DOX-EDT-IONPs were found to be effective in apoptotic-induced GBM cell death (over 90%) within 48 h of treatment. Gene expression studies revealed a significant downregulation of TOP II and Ku70, crucial enzymes for DNA repair and replication, as well as MiR-155 oncogene, concomitant with an upregulation of caspase 3 and tumor suppressors i.e., p53, MEG3 and GAS5, in U251 cells upon treatment with DOX-EDT-IONPs. An in vitro MDCK-MDR1-GBM co-culture model was used to assess the BBB permeability and anti-tumor activity of the DOX-EDT-IONPs and DOX treatments. While DOX-EDT-IONP showed improved permeability of DOX across MDCK-MDR1 monolayers compared to DOX alone, cytotoxicity in U251 cells was similar in both treatment groups. Using a cadherin binding peptide (ADTC5) to transiently open tight junctions, in combination with an external magnetic field, significantly enhanced both DOX-EDT-IONP permeability and cytotoxicity in the MDCK-MDR1-GBM co-culture model. Therefore, the combination of magnetic enhanced convective diffusion and the cadherin binding peptide for transiently opening the BBB tight junctions are expected to enhance the efficacy of GBM chemotherapy using the DOX-EDT-IONPs. In general, the developed approach enables the chemotherapeutic to overcome both BBB and multidrug resistance (MDR) glioma cells while providing site-specific magnetic targeting.
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http://dx.doi.org/10.1038/s41598-020-68017-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347880PMC
July 2020

Salinomycin-Loaded Iron Oxide Nanoparticles for Glioblastoma Therapy.

Nanomaterials (Basel) 2020 Mar 6;10(3). Epub 2020 Mar 6.

Department of Biomedical Engineering, University of Manitoba, Winnipeg, MB R3T 5V6, Canada.

Salinomycin is an antibiotic introduced recently as a new and effective anticancer drug. In this study, magnetic iron oxide nanoparticles (IONPs) were utilized as a drug carrier for salinomycin for potential use in glioblastoma (GBM) chemotherapy. The biocompatible polyethylenimine (PEI)-polyethylene glycol (PEG)-IONPs (PEI-PEG-IONPs) exhibited an efficient uptake in both mouse brain-derived microvessel endothelial (bEnd.3) and human U251 GBM cell lines. The salinomycin (Sali)-loaded PEI-PEG-IONPs (Sali-PEI-PEG-IONPs) released salinomycin over 4 days, with an initial release of 44% ± 3% that increased to 66% ± 5% in acidic pH. The Sali-IONPs inhibited U251 cell proliferation and decreased their viability (by approximately 70% within 48 h), and the nanoparticles were found to be effective in reactive oxygen species-mediated GBM cell death. Gene studies revealed significant activation of caspases in U251 cells upon treatment with Sali-IONPs. Furthermore, the upregulation of tumor suppressors (i.e., p53, Rbl2, Gas5) was observed, while TopII, Ku70, CyclinD1, and Wnt1 were concomitantly downregulated. When examined in an blood-brain barrier (BBB)-GBM co-culture model, Sali-IONPs had limited penetration (1.0% ± 0.08%) through the bEnd.3 monolayer and resulted in 60% viability of U251 cells. However, hyperosmotic disruption coupled with an applied external magnetic field significantly enhanced the permeability of Sali-IONPs across bEnd.3 monolayers (3.2% ± 0.1%) and reduced the viability of U251 cells to 38%. These findings suggest that Sali-IONPs combined with penetration enhancers, such as hyperosmotic mannitol and external magnetic fields, can potentially provide effective and site-specific magnetic targeting for GBM chemotherapy.
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http://dx.doi.org/10.3390/nano10030477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7153627PMC
March 2020

Acute upregulation of bone morphogenetic protein-4 regulates endogenous cell response and promotes cell death in spinal cord injury.

Exp Neurol 2020 03 24;325:113163. Epub 2019 Dec 24.

Department of Physiology and Pathophysiology, Regenerative Medicine Program, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, Manitoba, Canada; Children's Hospital Research Institute of Manitoba, Winnipeg, Manitoba, Canada. Electronic address:

Traumatic spinal cord injury (SCI) elicits a cascade of secondary injury mechanisms that induce profound changes in glia and neurons resulting in their activation, injury or cell death. The resultant imbalanced microenvironment of acute SCI also negatively impacts regenerative processes in the injured spinal cord. Thus, it is imperative to uncover endogenous mechanisms that drive these acute injury events. Here, we demonstrate that the active form of bone morphogenetic protein-4 (BMP4) is robustly and transiently upregulated in acute SCI in rats. BMP4 is a key morphogen in neurodevelopment; however, its role in SCI is not fully defined. Thus, we elucidated the ramification of BMP4 upregulation in a preclinical model of compressive/contusive SCI in the rat by employing noggin, an endogenous antagonist of BMP ligands, and LDN193189, an intracellular inhibitor of BMP signaling. In parallel, we studied cell-specific effects of BMP4 on neural precursor cells (NPCs), oligodendrocyte precursor cells (OPCs), neurons and astrocytes in vitro. We demonstrate that activation of BMP4 inhibits differentiation of spinal cord NPCs and OPCs into mature myelin-expressing oligodendrocytes, and acute blockade of BMPs promotes oligodendrogenesis, oligodendrocyte preservation and remyelination after SCI. Importantly, we report for the first time that BMP4 directly induces caspase-3 mediated apoptosis in neurons and oligodendrocytes in vitro, and noggin and LDN193189 remarkably attenuate caspase-3 activation and lipid peroxidation in acute SCI. BMP4 also enhances the production of inhibitory chondroitin sulfate proteoglycans (CSPGs) in activated astrocytes in vitro and after SCI. Interestingly, our work reveals that despite the beneficial effects of BMP inhibition in acute SCI, neither noggin nor LDN193189 treatment resulted in long-term functional recovery. Collectively, our findings suggest a role for BMP4 in regulating acute secondary injury mechanisms following SCI, and a potential target for combinatorial approaches to improve endogenous cell response and remyelination.
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http://dx.doi.org/10.1016/j.expneurol.2019.113163DOI Listing
March 2020

Simple, Hackable, Size-Selective, Amine-Functionalized Fe-Oxide Nanoparticles for Biomedical Applications.

Langmuir 2018 02 12;34(8):2748-2757. Epub 2018 Feb 12.

Department of Physics and Astronomy, University of Manitoba , Winnipeg, Manitoba R3T 2N2, Canada.

A facile one-pot method for synthesizing amine-functionalized nonspherical FeO nanoparticles in gram-scale quantities is presented using just a single source of iron (iron(II) chloride) and an amine (triethylamine). The amine not only transforms iron salt to FeO, but also directs the morphology of the nanoparticles along with the temperature of the reaction and functionalizes them, making the synthesis very economical. By modifying the surface further, these nanoparticles promise to offer useful biomedical applications. For example, after biocide coating, the particles are found to be 100% effective in deactivating methicillin-resistant Staphylococcus aureus (MRSA) bacteria in 2 h. Cellular-uptake studies using biocompatible EDTA-Na (N-(trimethoxysilyl-propyl)ethylenediaminetriacetate, trisodium salt)-coated nanoparticles in human glioblastoma U-251 cells show that the majority of the particles are internalized by the cells in the presence of a small dc-magnetic field, making these particles a potential candidate as drug carriers for magnetic field-targeted delivery and hyperthermia.
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http://dx.doi.org/10.1021/acs.langmuir.7b02822DOI Listing
February 2018

Neuregulin-1 promotes remyelination and fosters a pro-regenerative inflammatory response in focal demyelinating lesions of the spinal cord.

Glia 2018 03 17;66(3):538-561. Epub 2017 Nov 17.

Regenerative Medicine Program, Department of Physiology and Pathophysiology, Spinal Cord Research Centre, Rady Faculty of Health Sciences University of Manitoba, Winnipeg, Manitoba, Canada.

Oligodendroglial cell death and demyelination are hallmarks of neurotrauma and multiple sclerosis that cause axonal damage and functional impairments. Remyelination remains a challenge as the ability of endogenous precursor cells for oligodendrocyte replacement is hindered in the unfavorable milieu of demyelinating conditions. Here, in a rat model of lysolecithin lysophosphatidyl-choline (LPC)-induced focal demyelination, we report that Neuregulin-1 (Nrg-1), an important factor for oligodendrocytes and myelination, is dysregulated in demyelinating lesions and its bio-availability can promote oligodendrogenesis and remyelination. We delivered recombinant human Nrg-1β1 (rhNrg-1β1) intraspinally in the vicinity of LPC demyelinating lesion in a sustained manner using poly lactic-co-glycolic acid microcarriers. Availability of Nrg-1 promoted generation and maturation of new oligodendrocytes, and accelerated endogenous remyelination by both oligodendrocyte and Schwann cell populations in demyelinating foci. Importantly, Nrg-1 enhanced myelin thickness in newly remyelinated spinal cord axons. Our complementary in vitro studies also provided direct evidence that Nrg-1 significantly promotes maturation of new oligodendrocytes and facilitates their transition to a myelinating phenotype. Nrg-1 therapy remarkably attenuated the upregulated expression chondroitin sulfate proteoglycans (CSPGs) specific glycosaminoglycans in the extracellular matrix of demyelinating foci and promoted interleukin-10 (IL-10) production by immune cells. CSPGs and IL-10 are known to negatively and positively regulate remyelination, respectively. We found that Nrg-1 effects are mediated through ErbB2 and ErbB4 receptor activation. Our work provides novel evidence that dysregulated levels of Nrg-1 in demyelinating lesions of the spinal cord pose a challenge to endogenous remyelination, and appear to be an underlying cause of myelin thinning in newly remyelinated axons.
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http://dx.doi.org/10.1002/glia.23264DOI Listing
March 2018

Perturbation of redox balance after thioredoxin reductase deficiency interrupts autophagy-lysosomal degradation pathway and enhances cell death in nutritionally stressed SH-SY5Y cells.

Free Radic Biol Med 2016 12 28;101:53-70. Epub 2016 Sep 28.

Spinal Cord Research Center, Department of Physiology and Pathophysiology, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada; Regenerative Medicine Program, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada.

Oxidative damage and aggregation of cellular proteins is a hallmark of neuronal cell death after neurotrauma and chronic neurodegenerative conditions. Autophagy and ubiquitin protease system are involved in degradation of protein aggregates, and interruption of their function is linked to apoptotic cell death in these diseases. Oxidative modification of cysteine groups in key molecular proteins has been linked to modification of cellular systems and cell death in these conditions. Glutathione and thioredoxin systems provide reducing protons that can effectively reverse protein modifications and promote cell survival. The central role of Thioredoxin in inhibition of apoptosis is well identified. Additionally, its involvement in initiation of autophagy has been suggested recently. We therefore aimed to investigate the involvement of Thioredoxin system in autophagy-apoptosis processes. A model of serum deprivation in SH-SY5Y was used that is associated with autophagy and apoptosis. Using pharmacological and RNA-editing technology we show that Thioredoxin reductase deficiency in this model enhances oxidative stress and interrupts the early protective autophagy and promotes apoptosis. This was associated with decreased protein-degradation in lysosomes due to altered lysosomal acidification and accumulation of autophagosomes as well as impairment in proteasome pathway. We further confirmed that the extent of oxidative stress is a determining factor in autophagy- apoptosis interplay, as upregulation of cellular reducing capacity by N-acetylcysteine prevented impairment in autophagy and proteasome systems thus promoted cell viability. Our study provides evidence that excessive oxidative stress inhibits protein degradation systems and affects the final stages of autophagy by inhibiting autolysosome maturation: a novel mechanistic link between protein aggregation and conversion of autophagy to apoptosis that can be applicable to neurodegenerative diseases.
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http://dx.doi.org/10.1016/j.freeradbiomed.2016.09.026DOI Listing
December 2016

Differential internalization of brick shaped iron oxide nanoparticles by endothelial cells.

J Mater Chem B 2016 Sep 19;4(35):5913-5920. Epub 2016 Aug 19.

Department of Pharmacology and Therapeutics, University of Manitoba, 710 William Avenue, Winnipeg, Manitoba R3E 0T6, Canada.

Nanoparticles targeting endothelial cells to treat diseases such as cancer, oxidative stress, and inflammation have traditionally relied on ligand-receptor based delivery. The present studies examined the influence of nanoparticle shape in regulating preferential uptake of nanoparticles in endothelial cells. Spherical and brick shaped iron oxide nanoparticles (IONPs) were synthesized with identical negatively charged surface coating. The nanobricks showed a significantly greater uptake profile in endothelial cells compared to nanospheres. Application of an external magnetic field significantly enhanced the uptake of nanobricks but not nanospheres. Transmission electron microscopy revealed differential internalization of nanobricks in endothelial cells compared to epithelial cells. Given the reduced uptake of nanobricks in endothelial cells treated with caveolin inhibitors, the increased expression of caveolin-1 in endothelial cells compared to epithelial cells, and the ability of IONP nanobricks to interfere with caveolae-mediated endocytosis process, a caveolae-mediated pathway is proposed as the mechanism for differential internalization of nanobricks in endothelial cells.
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http://dx.doi.org/10.1039/c6tb01480aDOI Listing
September 2016

Pelota Regulates Epidermal Differentiation by Modulating BMP and PI3K/AKT Signaling Pathways.

J Invest Dermatol 2016 08 7;136(8):1664-1671. Epub 2016 May 7.

Institute of Human Genetics, University of Göttingen, D-37073 Göttingen, Germany. Electronic address:

The depletion of evolutionarily conserved pelota protein causes impaired differentiation of embryonic and spermatogonial stem cells. In this study, we show that temporal deletion of pelota protein before epidermal barrier acquisition leads to neonatal lethality due to perturbations in permeability barrier formation. Further analysis indicated that this phenotype is a result of failed processing of profilaggrin into filaggrin monomers, which promotes the formation of a protective epidermal layer. Molecular analyses showed that pelota protein negatively regulates the activities of bone morphogenetic protein and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways in the epidermis. To address whether elevated activities of bone morphogenetic protein and PI3K/AKT signaling pathways were the cause for the perturbed epidermal barrier in Pelo-deficient mice, we made use of organotypic cultures of skin explants from control and mutant embryos at embryonic day 15.5. Inhibition of PI3K/AKT signaling did not significantly affect the bone morphogenetic protein activity. However, inhibition of bone morphogenetic protein signaling caused a significant attenuation of PI3K/AKT activity in mutant skin and, more interestingly, the restoration of profilaggrin processing and normal epidermal barrier function. Therefore, increased activity of the PI3K/AKT signaling pathway in Pelo-deficient skin might conflict with the dephosphorylation of profilaggrin and thereby affect its proper processing into filaggrin monomers and ultimately the epidermal differentiation.
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http://dx.doi.org/10.1016/j.jid.2016.04.020DOI Listing
August 2016

Biodistribution of negatively charged iron oxide nanoparticles (IONPs) in mice and enhanced brain delivery using lysophosphatidic acid (LPA).

Nanomedicine 2016 10 25;12(7):1775-1784. Epub 2016 Apr 25.

Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, MB, Canada. Electronic address:

Effective treatment of brain disorders requires a focus on improving drug permeability across the blood-brain barrier (BBB). Herein, we examined the pharmacokinetic properties of negatively charged iron oxide nanoparticles (IONPs) and the capability of using lysophosphatidic acid (LPA) to transiently disrupt the tight junctions and allow IONPs to enter the brain. Under normal conditions, IONPs had a plasma half-life of six minutes, with the liver and spleen being the major organs of deposition. Treatment with LPA enhanced accumulation of IONPs in the brain and spleen (approximately 4-fold vs. control). LPA and IONP treated mice revealed no sign of peripheral immune cell infiltration in the brain and no significant activation of microglia or astrocytes. These studies show improved delivery efficiency of IONPs following LPA administration. Our findings suggest transient disruption of the BBB may be a safe and effective method for increasing IONP delivery to the brain.
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http://dx.doi.org/10.1016/j.nano.2016.04.008DOI Listing
October 2016

Modulation of intercellular junctions by cyclic-ADT peptides as a method to reversibly increase blood-brain barrier permeability.

J Pharm Sci 2015 Mar 12;104(3):1065-75. Epub 2015 Jan 12.

Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas, 66047.

It is challenging to deliver molecules to the brain for diagnosis and treatment of brain diseases. This is primarily because of the presence of the blood-brain barrier (BBB), which restricts the entry of many molecules into the brain. In this study, cyclic-ADT peptides (ADTC1, ADTC5, and ADTC6) have been shown to modify the BBB to enhance the delivery of marker molecules [e.g., (14) C-mannitol, gadolinium-diethylenetriaminepentacetate (Gd-DTPA)] to the brain via the paracellular pathways of the BBB. The hypothesis is that these peptides modulate cadherin interactions in the adherens junctions of the vascular endothelial cells forming the BBB to increase paracellular drug permeation. In vitro studies indicated that ADTC5 had the best profile to inhibit adherens junction resealing in Madin-Darby canine kidney cell monolayers in a concentration-dependent manner (IC50 = 0.3 mM) with a maximal response at 0.4 mM. Under the current experimental conditions, ADTC5 improved the delivery of (14) C-mannitol to the brain about twofold compared with the negative control in the in situ rat brain perfusion model. Furthermore, ADTC5 peptide increased in vivo delivery of Gd-DTPA to the brain of Balb/c mice when administered intravenously. In conclusion, ADTC5 has the potential to improve delivery of diagnostic and therapeutic agents to the brain.
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http://dx.doi.org/10.1002/jps.24309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4442687PMC
March 2015

The FGF-2-triggered protection of cardiac subsarcolemmal mitochondria from calcium overload is mitochondrial connexin 43-dependent.

Cardiovasc Res 2014 Jul 20;103(1):72-80. Epub 2014 Mar 20.

Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Taché Avenue, Winnipeg, Manitoba, Canada R2H 2A6 Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada Department of Human Anatomy and Cell Sciences, University of Manitoba, Winnipeg, Manitoba, Canada

Aims: Fibroblast growth factor 2 (FGF-2) protects the heart from ischaemia- and reperfusion-induced cell death by a mechanism linked to protein kinase C (PKC)ε-mediated connexin 43 (Cx43) phosphorylation. Cx43 localizes predominantly to gap junctions, but has also been detected at subsarcolemmal (SSM), but not interfibrillar (IFM), mitochondria, where it is considered important for cardioprotection. We have now examined the effect of FGF-2 administration to the heart on resistance to calcium-induced permeability transition (mPTP) of isolated SSM vs. IFM suspensions, in relation to mitochondrial PKCε/Cx43 levels, phosphorylation, and the presence of peptide Gap27, a Cx43 channel blocker.

Methods And Results: FGF-2 perfusion increased resistance to calcium-induced mPTP in SSM and IFM suspensions by 2.9- and 1.7-fold, respectively, compared with their counterparts from vehicle-perfused hearts, assessed spectrophotometrically as cyclosporine A-inhibitable swelling. The salutary effect of FGF-2 was lost in SSM, but not in IFM, in the presence of Gap27. FGF-2 perfusion increased relative levels of PKCε, phospho(p) PKCε, and Tom-20 translocase in SSM and IFM, and of Cx43 in SSM. Phospho-serine (pS) 262- and pS368-Cx43 showed a 30- and 8-fold increase, respectively, in SSM from FGF-2-treated, compared with untreated, hearts. Stimulation of control SSM with phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased both calcium tolerance and mitochondrial Cx43 phosphorylation at S262 and S368. The PMA-induced phosphorylation of mitochondrial Cx43 was prevented by εV1-2, a PKCε-inhibiting peptide.

Conclusions: SSM are more responsive than IFM to FGF-2-triggered protection from calcium-induced mPTP, by a mitochondrial Cx43 channel-mediated pathway, associated with mitochondrial Cx43 phosphorylation at PKCε target sites.
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http://dx.doi.org/10.1093/cvr/cvu066DOI Listing
July 2014

Caspase 3 activity in isolated fetal rat lung fibroblasts and rat periodontal ligament fibroblasts: cigarette smoke induced alterations.

Tob Induc Dis 2013 Dec 6;11(1):25. Epub 2013 Dec 6.

Departments of Oral Biology, University of Manitoba and Manitoba Institute for Child Health, Winnipeg, Canada.

Background: Cigarette smoking is the leading cause of preventable death and has been implicated in pathogenesis of pulmonary, oral and systemic diseases. Smoking during pregnancy is a risk factor for the developing fetus and may be a major cause of infant mortality. Moreover, the oral cavity, and all cells within are the first to be exposed to cigarette smoke and may be a possible source for the spread of toxins to other organs of the body. Fibroblasts in general are morphologically heterogeneous connective tissue cells with diverse functions. Apoptosis or programmed cell death is a crucial process during embryogenesis and for the maintenance of homeostasis throughout life. Deregulation of apoptosis has been implicated in abnormal lung development in the fetus and disease progression in adults. Caspases are proteases which belong to the family of cysteine aspartic acid proteases and are key components for downstream amplification of intracellular apoptotic signals. Of 14 known caspases, caspase-3 is the key executioner of apoptosis. In the present study we explored the hypothesis that cigarette smoke (CS) extract activates caspase-3 in two types of fibroblasts, both of which would be exposed directly to cigarette smoke, isolated fetal rat lung fibroblasts and adult rat periodontal ligament (PDL) fibroblasts.

Methods: Isolated fetal rat lung fibroblasts and adult PDLs were used. Cells were exposed to different concentrations of CS for 60 min. Caspase-3 activity and its inhibition by Z-VAD-fmk were measured by caspase-3 fluorometric assay. The effect of CSE on cellular viability was measured using the MTT formazan assay. Caspase-3 expression was detected by western blot analysis and cellular localization of caspase-3 was determined by immunofluorescence using fluorescence microscopy.

Results: It was observed in fetal rat lung fibroblast cells that CSE extract significantly (p<0.05) increased caspase-3 activity and decrease cell proliferation. However, no significant changes in activity or viability were observed in PDLs.

Conclusions: This indicates CS activates caspase-3 the key regulatory point in apoptosis in fetal rat lung fibroblast cells suggesting that smoking during pregnancy may alter the developmental program of fetal lung, jeopardizing the establishment of critical cellular mechanisms necessary to expedite pulmonary maturation at birth.of critical cellular mechanisms necessary to expedite pulmonary maturation at birth.
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http://dx.doi.org/10.1186/1617-9625-11-25DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4029472PMC
December 2013

One-pot synthesis of iron oxide nanoparticles with functional silane shells: a versatile general precursor for conjugations and biomedical applications.

Langmuir 2013 Aug 16;29(34):10850-8. Epub 2013 Aug 16.

Department of Chemistry, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada.

Iron oxide nanoparticles (IONPs) and their surface modifications with therapeutic or diagnostic (theranostic, TN) agents are of great interest. Here we present a novel one-pot synthesis of a versatile general TN precursor (aminosilane-coated IONPs [IONP-Sil(NH2)]) with surface amine groups. Surface functional group conversion to carboxylic acid was accomplished by conjugating poly(ethylene glycol) diacid to IONP-Sil(NH2). The NPs were characterized using powder X-ray diffraction (XRD), transmission electron microscopy (TEM), high-resolution TEM, selected area electron diffraction (SAED), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared (FT-IR) spectroscopy. Biocompatibility and cell uptake profile of the nanoparticles were evaluated in-vitro using cultured liver cells (HepG2). Oleylamine (hydrophobic) and bovine serum albumin (BSA) as model drugs were attached to IONP-Sil-PEG(COOH). The ability of IONP-Sil(NH2) to bind small interfering RNA (siRNA) is also shown.
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http://dx.doi.org/10.1021/la402007dDOI Listing
August 2013

Characterization of cellular uptake and toxicity of aminosilane-coated iron oxide nanoparticles with different charges in central nervous system-relevant cell culture models.

Int J Nanomedicine 2013 6;8:961-70. Epub 2013 Mar 6.

Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Manitoba, Canada.

Background: Aminosilane-coated iron oxide nanoparticles (AmS-IONPs) have been widely used in constructing complex and multifunctional drug delivery systems. However, the biocompatibility and uptake characteristics of AmS-IONPs in central nervous system (CNS)-relevant cells are unknown. The purpose of this study was to determine the effect of surface charge and magnetic field on toxicity and uptake of AmS-IONPs in CNS-relevant cell types.

Methods: The toxicity and uptake profile of positively charged AmS-IONPs and negatively charged COOH-AmS-IONPs of similar size were examined using a mouse brain microvessel endothelial cell line (bEnd.3) and primary cultured mouse astrocytes and neurons. Cell accumulation of IONPs was examined using the ferrozine assay, and cytotoxicity was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

Results: No toxicity was observed in bEnd.3 cells at concentrations up to 200 μg/mL for either AmS-IONPs or COOH-AmS-IONPs. AmS-IONPs at concentrations above 200 μg/mL reduced neuron viability by 50% in the presence or absence of a magnetic field, while only 20% reductions in viability were observed with COOH-AmS-IONPs. Similar concentrations of AmS-IONPs in astrocyte cultures reduced viability to 75% but only in the presence of a magnetic field, while exposure to COOH-AmS-IONPs reduced viability to 65% and 35% in the absence and presence of a magnetic field, respectively. Cellular accumulation of AmS-IONPs was greater in all cell types examined compared to COOH-AmS-IONPs. Rank order of cellular uptake for AmS-IONPs was astrocytes > bEnd.3 > neurons. Accumulation of COOH-AmS-IONPs was minimal and similar in magnitude in different cell types. Magnetic field exposure enhanced cellular accumulation of both AmS- and COOH-AmS-IONPs.

Conclusion: Both IONP compositions were nontoxic at concentrations below 100 μg/mL in all cell types examined. At doses above 100 μg/mL, neurons were more sensitive to AmS-IONPs, whereas astrocytes were more vulnerable toward COOH-AmS-IONPs. Toxicity appears to be dependent on the surface coating as opposed to the amount of iron-oxide present in the cell.
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http://dx.doi.org/10.2147/IJN.S39048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3593762PMC
January 2014

A tea/vanadate decoction delivered orally over 14 months to diabetic rats induces long-term glycemic stability without organ toxicity.

Metabolism 2012 May 5;61(5):742-53. Epub 2011 Dec 5.

Department of Surgery, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R3E OW3.

Vanadium can induce potent hypoglycemic effects in type 1 and type 2 diabetes mellitus animals, but toxic adverse effects have inhibited the translation of these findings. Administration of vanadate in a black tea decoction has shown impressive hypoglycemic effects without evidence of toxicity in short-term studies. The purpose of this study was to investigate the hypoglycemic action and the toxic adverse effects of a tea/vanadate (T/V) decoction in diabetic rats over a 14-month treatment period. Streptozotocin-induced type 1 diabetes mellitus rats were orally gavaged with 40 mg sodium vanadate in a black tea decoction only when blood glucose levels were greater than 10 mmol/L. Glycemic status and liver and kidney function were monitored over 14 months. All of the diabetic rats in this treatment group (n = 25) required treatment with the T/V decoction at the start of the study to reduce blood glucose levels to less than 10 mmol/L. Diarrhea was uncommon among the T/V-treated animals during the first week of T/V treatment and was absent thereafter. There was no evidence of liver or kidney dysfunction or injury. From 2 to 6 months, fewer animals required the T/V treatment to maintain their blood glucose levels. After 9 months of treatment, none of the diabetic animals required any T/V to maintain their blood glucose levels at less than 10 mmol/L. Oral administration of a T/V decoction provides safe, long-acting hypoglycemic effects in type 1 diabetes mellitus rats. The typical glycemic signs of diabetes were absent for the last 5 months of the study.
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http://dx.doi.org/10.1016/j.metabol.2011.10.007DOI Listing
May 2012

Hyaluronidase 3 (HYAL3) knockout mice do not display evidence of hyaluronan accumulation.

Matrix Biol 2008 Oct 14;27(8):653-60. Epub 2008 Aug 14.

Department of Biochemistry and Medical Genetics, The University of Manitoba, Winnipeg, MB, Canada.

Hyaluronidases are endoglycosidases that initiate the breakdown of hyaluronan (HA), an abundant component of the vertebrate extracellular matrix. In humans, six paralogous genes encoding hyaluronidase-like sequences have been identified on human chromosomes 3p21.3 (HYAL2-HYAL1-HYAL3) and 7q31.3 (SPAM1-HYAL4-HYALP1). Mutations in one of these genes, HYAL1, were reported in a patient with mucopolysaccharidosis (MPS) IX. Despite the broad distribution of HA, the HYAL1-deficient patient exhibited a mild phenotype, suggesting other hyaluronidase family members contribute to constitutive HA degradation. Hyal3 knockout (Hyal3-/-) mice were generated to determine if HYAL3 had a role in constitutive HA degradation. Hyal3-/- mice were viable, fertile, and exhibited no gross phenotypic changes. X-ray analysis, histological studies of joints, whole-body weights, organ weights and the serum HA levels of Hyal3-/- mice were normal. No evidence of glycosaminoglycan accumulation, including vacuolization, was identified in the Hyal3-/- tissues analyzed. Remarkably, the only difference identified in Hyal3-/- mice was a subtle change in the alveolar structure and extracellular matrix thickness in lung-tissue sections at 12-14 months-of-age. We conclude that HYAL3 does not play a major role in constitutive HA degradation.
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http://dx.doi.org/10.1016/j.matbio.2008.07.006DOI Listing
October 2008

Caveolae facilitate muscarinic receptor-mediated intracellular Ca2+ mobilization and contraction in airway smooth muscle.

Am J Physiol Lung Cell Mol Physiol 2007 Dec 21;293(6):L1406-18. Epub 2007 Sep 21.

Department of Physiology, University of Manitoba, John Buhler Research Centre, Winnipeg, Manitoba, Canada.

Contractile responses of airway smooth muscle (ASM) determine airway resistance in health and disease. Caveolae microdomains in the plasma membrane are marked by caveolin proteins and are abundant in contractile smooth muscle in association with nanospaces involved in Ca(2+) homeostasis. Caveolin-1 can modulate localization and activity of signaling proteins, including trimeric G proteins, via a scaffolding domain. We investigated the role of caveolae in contraction and intracellular Ca(2+) ([Ca(2+)](i)) mobilization of ASM induced by the physiological muscarinic receptor agonist, acetylcholine (ACh). Human and canine ASM tissues and cells predominantly express caveolin-1. Muscarinic M(3) receptors (M(3)R) and Galpha(q/11) cofractionate with caveolin-1-rich membranes of ASM tissue. Caveolae disruption with beta-cyclodextrin in canine tracheal strips reduced sensitivity but not maximum isometric force induced by ACh. In fura-2-loaded canine and human ASM cells, exposure to methyl-beta-cyclodextrin (mbetaCD) reduced sensitivity but not maximum [Ca(2+)](i) induced by ACh. In contrast, both parameters were reduced for the partial muscarinic agonist, pilocarpine. Fluorescence microscopy revealed that mbetaCD disrupted the colocalization of caveolae-1 and M(3)R, but [N-methyl-(3)H]scopolamine receptor-binding assay revealed no effect on muscarinic receptor availability or affinity. To dissect the role of caveolin-1 in ACh-induced [Ca(2+)](i) flux, we disrupted its binding to signaling proteins using either a cell-permeable caveolin-1 scaffolding domain peptide mimetic or by small interfering RNA knockdown. Similar to the effects of mbetaCD, direct targeting of caveolin-1 reduced sensitivity to ACh, but maximum [Ca(2+)](i) mobilization was unaffected. These results indicate caveolae and caveolin-1 facilitate [Ca(2+)](i) mobilization leading to ASM contraction induced by submaximal concentrations of ACh.
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http://dx.doi.org/10.1152/ajplung.00312.2007DOI Listing
December 2007

Effect of NQO1 induction on the antitumor activity of RH1 in human tumors in vitro and in vivo.

Cancer Chemother Pharmacol 2005 Sep 5;56(3):307-16. Epub 2005 May 5.

Manitoba Institute of Cell Biology, CancerCare Manitoba, University of Manitoba, 675 McDermot Avenue, Winnipeg, Manitoba, R3E 0V9, Canada.

NQO1 is a reductive enzyme that is important for the activation of many bioreductive agents and is a target for an enzyme-directed approach to cancer therapy. It can be selectively induced in many tumor types by a number of compounds including dimethyl fumarate and sulforaphane. Mitomycin C is a bioreductive agent that is used clinically for treatment of solid tumors. RH1 (2,5-diaziridinyl-3-(hydroxymethyl)- 6-methyl-1,4-benzoquinone) is a new bioreductive agent currently in clinical trials. We have shown previously that induction of NQO1 can enhance the antitumor activity of mitomycin C in tumor cells in vitro and in vivo. As RH1 is activated selectively by NQO1 while mitomycin C is activated by many reductive enzymes, we investigated whether induction of NQO1 would produce a greater enhancement of the antitumor activity of RH1 compared with mitomycin C. HCT116 human colon cancer cells and T47D human breast cancer cells were incubated with or without dimethyl fumarate or sulforaphane followed by mitomycin C or RH1 treatment, and cytotoxic activity was measured by a clonogenic (HCT116) or MTT assay (T47D). Dimethyl fumarate and sulforaphane treatment increased NQO1 activity by 1.4- to 2.8-fold and resulted in a significant enhancement of the antitumor activity of mitomycin C, but not of RH1. This appeared to be due to the presence of a sufficient constitutive level of NQO1 activity in the tumor cells to fully activate the RH1. Mice were implanted with HL60 human promyelocytic leukemia cells, which have low levels of NQO1 activity. The mice were fed control or dimethyl fumarate-containing diet and were treated with RH1. NQO1 activity in the tumors increased but RH1 produced no antitumor activity in mice fed control or dimethyl fumarate diet. This is consistent with a narrow window of NQO1 activity between no RH1 activation and maximum RH1 activation. This study suggests that selective induction of NQO1 in tumor cells is not likely to be an effective strategy for enhancing the antitumor activity of RH1. In addition, we found that RH1 treatment produced significant leukopenia in mice that may be of concern in the clinic. These results suggest that the ease of reduction of RH1 by NQO1 makes it a poor candidate for an enzyme-directed approach to cancer therapy.
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http://dx.doi.org/10.1007/s00280-004-0961-4DOI Listing
September 2005

A comparison of biologically variable ventilation to recruitment manoeuvres in a porcine model of acute lung injury.

Respir Res 2004 Nov 24;5:22. Epub 2004 Nov 24.

Department of Anaesthesia, University of Manitoba, Winnipeg, Manitoba, Canada.

Background: Biologically variable ventilation (return of physiological variability in rate and tidal volume using a computer-controller) was compared to control mode ventilation with and without a recruitment manoeuvre - 40 cm H2O for 40 sec performed hourly; in a porcine oleic acid acute lung injury model.

Methods: We compared gas exchange, respiratory mechanics, and measured bronchoalveolar fluid for inflammatory cytokines, cell counts and surfactant function. Lung injury was scored by light microscopy. Pigs received mechanical ventilation (FIO2 = 0.3; PEEP 5 cm H2O) in control mode until PaO2 decreased to 60 mm Hg with oleic acid infusion (PaO2/FIO2 <200 mm Hg). Additional PEEP to 10 cm H2O was added after injury. Animals were randomized to one of the 3 modes of ventilation and followed for 5 hr after injury.

Results: PaO2 and respiratory system compliance was significantly greater with biologically variable ventilation compared to the other 2 groups. Mean and mean peak airway pressures were also lower. There were no differences in cell counts in bronchoalveolar fluid by flow cytometry, or interleukin-8 and -10 levels between groups. Lung injury scoring revealed no difference between groups in the regions examined. No differences in surfactant function were seen between groups by capillary surfactometry.

Conclusions: In this porcine model of acute lung injury, various indices to measure injury or inflammation did not differ between the 3 approaches to ventilation. However, when using a low tidal volume strategy with moderate levels of PEEP, sustained improvements in arterial oxygen tension and respiratory system compliance were only seen with BVV when compared to CMV or CMV with a recruitment manoeuvre.
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http://dx.doi.org/10.1186/1465-9921-5-22DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC535805PMC
November 2004

Gonadal dysgenesis with X-monosomy in a cynomolgus monkey (Macaca fascicularis).

Am J Primatol 1990 ;22(1):51-59

Primate Research Institute, New Mexico State University, Holloman AFB, New Mexico.

A 3-year-old female cynomolgus monkey (Macaca fascicularis) was found to have inappropriately high circulating immunoassayable follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels compatible with primary gonadal failure. Hypergonadotropic hypogonadism was confirmed by the findings of persistently high serum gonadotropin levels by both LH bioassay and FSH and LH radioimmunoassays and low levels of serum estradiol. In addition, circhoral gonadotropin pulsatility and an exaggerated response to a gonadotropin-releasing hormone challenge were demonstrated. Both FSH and LH coeluted in a single peak on gel filtration column chromatography. Clinically, the animal showed the following features of Turner syndrome: small body size, sexual underdevelopment, gonadal streaks with absent follicles, and a chromosomal constitution of 41,X. A similar case has been reported previously in a rhesus monkey (Macaca mulatta). Considering the fetal and live birth prevalences in humans of aneuploidy in general and X-monosomy in particular, one would predict that this chromosomal aberration underlies a high number of pregnancy failures in nonhuman primates.
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http://dx.doi.org/10.1002/ajp.1350220103DOI Listing
January 1990