University of Canterbury Home
    • Admin
    UC Research Repository
    UC Library
    JavaScript is disabled for your browser. Some features of this site may not work without it.
    View Item 
    1. UC Home
    2. Library
    3. UC Research Repository
    4. Faculty of Science | Te Kaupeka Pūtaiao
    5. Science: Theses and Dissertations
    6. View Item
    1. UC Home
    2.  > 
    3. Library
    4.  > 
    5. UC Research Repository
    6.  > 
    7. Faculty of Science | Te Kaupeka Pūtaiao
    8.  > 
    9. Science: Theses and Dissertations
    10.  > 
    11. View Item

    Low density lipoprotein induction of intracellular oxidants production (2015)

    Thumbnail
    View/Open
    thesis_fulltext.pdf (9.966Mb)
    Type of Content
    Theses / Dissertations
    UC Permalink
    http://hdl.handle.net/10092/11300
    http://dx.doi.org/10.26021/5996
    
    Thesis Discipline
    Biochemistry
    Degree Name
    Doctor of Philosophy
    Publisher
    University of Canterbury. School of Biological Sciences
    Collections
    • Science: Theses and Dissertations [4655]
    Authors
    Othman, Mohd Izani
    show all
    Abstract

    Atherosclerosis is a complex cardiovascular disease characterized by chronic progressive inflammation of the arteries. The progression of atherosclerosis from fatty streak to advance atherosclerotic plaque involves the development of a necrotic core region consists of cholesterol, lipids, calcium (Ca²⁺), dead cells and other cellular debris. Macrophage infiltrations occurred in all stages of atherosclerotic progression and they are abundantly found in atherosclerotic plaques. Oxidised low density lipoprotein (oxLDL) plays a vital role in the initiation and development of atherosclerosis. OxLDL is present within atherosclerotic plaque and has been shown to be cytotoxic to various types of cells including macrophages. This research initially examined the cytotoxic effects of copper oxidised LDL on U937, human monocytes and HMDM cells. As expected oxLDL was cytotoxic; causing rapid, concentration and time dependent cell viability loss in all types of cells examined. Examination of the cell morphology showed that oxLDL caused a necrotic like cell death characterised by cell swelling and lysis. Flow cytometric assay coupled with propidium iodide (PI) staining of necrotic cells was compared to MTT reduction assays of cell viability. The flow cytometric technique had the advantage over the MTT reduction assay of being rapid and showing both the live and dead cell levels at an individual cell level. The progression of oxLDL-induced cell death correlated with the rapid increased in intracellular ROS production in the cytosol and the mitochondria. Immunoblotting results showed that oxLDL induced NADPH oxidase (NOX) activation and increased p47phox expression. This suggests NOX as the generator of reactive oxygen species (ROS) induced by oxLDL in these cells. However, apocynin and VAS2870, the two NOX inhibitors used in this study, were unable to inhibit the ROS generation caused by the oxLDL. This suggests that either these inhibitors are unable to inhibit the targeted NOX or other sources of ROS might exist and contributed to the overall increase in oxidative flux. OxLDL caused a rapid increase in cytosolic Ca²⁺ level. This was contributed by the extracellular Ca²⁺ source as well as Ca²⁺ mobilisation from the intracellular stores such as endoplasmic reticulum (ER). OxLDL-induced intracellular Ca²⁺ increase also correlated with the increase in intracellular ROS. Nevertheless, blocking of oxLDL-induced intracellular Ca²⁺ elevation by Ca²⁺ chelator, EGTA, did not reduce intracellular ROS generation. Accordingly, this suggests that oxLDL-induced intracellular Ca²⁺ increase is not the cause for oxLDL-induced cell death. Additionally, oxLDL may also initiate a Ca²⁺-independent cell death pathway. The excess cytosolic Ca²⁺ taken up by the mitochondria may be detrimental and could result in mitochondrial Ca²⁺ overload. This will increase mitochondrial ROS production and initiate mitochondrial permeability transition (MPT) pores opening. Consequently, this could collapse the mitochondrial membrane potential (𝚫𝚿m) due to the rupture of outer mitochondrial membrane (OMM) and resulted in cell death. Blocking of Ca²⁺ uptake into the mitochondria by ruthenium red protected cells from oxLDL-mediated cell death, possibly by reducing ROS production and preventing MPT activation. This study also demonstrated the protective effect of 7,8-dihydroneopterin (7,8-NP) on oxLDL-induced oxidative stress. 7,8-NP protected cells from oxLDL-induced intracellular ROS generation and cell viability loss. Intracellular Ca²⁺ increase was also reduced by 7,8-NP in particular after 3 hours incubation with oxLDL. The action of 7,8-NP was better than that of apocynin in protecting U937 cells from oxLDL suggests its potential ability to scavenge ROS from various sources. Studies have implicated the involvement of H₂S in various biological processes including atherosclerosis. Thus, the disruption of H₂S homeostasis may contribute to the progression of atherosclerosis. Slow releasing H₂S molecules (H₂S donors) have been developed for a controlled and stable delivery of H₂S to cells. In this study, specific H₂S donors, including one which targets the mitochondria, were found to be protective against oxLDL-induced cell death in U937, human monocytes and HMDM cells. Although the exact mechanism is yet to be elucidated, these H₂S donors were shown to block the elevation of intracellular Ca²⁺ and ROS production mediated by oxLDL. Therefore, these H₂S donors could be the potential candidates for future development of therapeutics in treating atherosclerosis.

    Keywords
    oxLDL; atherosclerosis; reactive oxygen species; calcium
    Rights
    Copyright Mohd Izani Othman
    https://canterbury.libguides.com/rights/theses

    Related items

    Showing items related by title, author, creator and subject.

    • Role of Intracellular Oxidant Release in Oxidised Low Lipoprotein - Induced U937 Cell Death 

      Chen, Alpha Yan (University of Canterbury. School of Biological Science, 2012)
      Atherosclerosis is a complex inflammation condition involving the accumulation of lipid-filled macrophages within the artery wall. Progression of the initial fatty streak to an advanced atherosclerotic plaque is characterized ...
    • Identification of the kinase cascades mediating oxidised low density lipoprotein-induced intracellular oxidative stress. 

      Steyn, Nina (University of Canterbury, 2018)
      The oxidation of low density lipoprotein (LDL) to form cytotoxic oxidised LDL (oxLDL) is a central atherogenic process. Exposure to oxLDL triggers an oxidative stress response in various immune and vascular cell types ...
    • 7,8-Dihydroneopterin-mediated protection of low density lipoprotein, but not human macrophages, from oxidative stress 

      Firth, Carole Anne (University of Canterbury. Biological Sciences, 2006)
      Any lipoproteins and cells present in the inflammatory environment of atherosclerotic plaques are likely to be exposed to high levels of oxidative stress. As 7,8-dihydroneopterin (7,8-NP) is synthesized by interferon-γ ...
    Advanced Search

    Browse

    All of the RepositoryCommunities & CollectionsBy Issue DateAuthorsTitlesSubjectsThesis DisciplineThis CollectionBy Issue DateAuthorsTitlesSubjectsThesis Discipline

    Statistics

    View Usage Statistics
    • SUBMISSIONS
    • Research Outputs
    • UC Theses
    • CONTACTS
    • Send Feedback
    • +64 3 369 3853
    • ucresearchrepository@canterbury.ac.nz
    • ABOUT
    • UC Research Repository Guide
    • Copyright and Disclaimer
    • SUBMISSIONS
    • Research Outputs
    • UC Theses
    • CONTACTS
    • Send Feedback
    • +64 3 369 3853
    • ucresearchrepository@canterbury.ac.nz
    • ABOUT
    • UC Research Repository Guide
    • Copyright and Disclaimer