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Thesis-Dec-2023-Hines9.87 MB
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Dissertation
Lipid oxidation links mitochondria to the redox environment and articular joint physiology
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Autumn 2023
DOI: 10.25820/etd.006847
Abstract
Lipid oxidation is thought to contribute to the initiation and progression of osteoarthritis (OA). Previous data from our research group have agreed with other basic science observations showing lipid antioxidants like α-tocopherol prevent mitochondrial loss after mechanical injury. However, α-tocopherol supplementation failed to translate clinically, and higher serum concentrations were associated with more severe radiographic indications of OA. This disconnect between basic research and clinical outcomes indicated a need for more thorough investigation of lipid oxidation within chondrocyte redox physiology. This thesis is an investigation of the role of lipid oxidation in articular chondrocytes using rigorous redox focused methods. To test the effects of modulating lipid oxidation on articular chondrocytes, osteochondral explants were dosed with increasing concentrations of α-tocopherol and mitochondrial content was evaluated. This study demonstrated mitochondrial content increased upon treatment with α-tocopherol in a dose-dependent manner. Promoting lipid oxidation with tert-butyl hydroperoxide decreased mitochondrial content. This decrease in mitochondrial content was also accompanied by a decrease in lipid oxidation, suggesting increasing lipid oxidation decreased function or decreased mitochondria and created a more reduced redox environment.
Next, we wanted to investigate how endogenous oxidants were broadly related to lipid oxidation. First, we adapted immuno-spin trapping for use in articular cartilage. This technique detects radicals on biomacromolecules providing an indication of global oxidative damage independent of radical species. We pretreated osteochondral explants with α-tocopherol before delivering a well characterized impact to generate mitochondrial oxidant production. We observed an increase in radical positive cells with impact that decreased with treatment with α-tocopherol before impact. However, α-tocopherol without impact increased average radical positive cells above controls, suggesting that decreasing lipid oxidation not only increased mitochondrial content but also increased basal oxidant production, broadly.
Finally, to establish that this pathway might have important physiological effects in the absence of injury, the effect of transgenic overexpression of glutathione peroxidase 4 (TgGPx4), a lipid antioxidant enzyme, was evaluated during aging. Young TgGPx4 mice had increased mitochondrial content compared to WT. Interestingly, except for slightly increased 4-hydroxynonenal modified proteins the young TgGPx4 redox markers were comparable to the WT. This suggests that, despite the difference in mitochondrial content, most features of the redox environment are maintained in vivo in young mice. However aged (18+ months) TgGPx4 mice had high variability in mitochondrial content both above and below the WT average. The high variability in mitochondrial content was echoed in the high variability of the redox markers compared to controls as well as other key molecular marks. This suggests that chronic TgGPx4 disrupted lipid peroxidation pathways crucial for redox metabolic regulation during aging.
Based on these findings, we conclude that lipid oxidation regulates chondrocyte mitochondria dynamics to protect against irreversible oxidative damage. This complex mechanism may explain the disconnect between basic research and the lack of clinical translation, while providing the initial observations for a new theory of chondrocyte redox metabolism.
Details
- Title: Subtitle
- Lipid oxidation links mitochondria to the redox environment and articular joint physiology
- Creators
- Madeline R Hines
- Contributors
- Mitchell C Coleman (Advisor)Garry R Buettner (Committee Member)Jessica E Goetz (Committee Member)David K Meyerholz (Committee Member)Douglas R Spitz (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Free Radical and Radiation Biology
- Date degree season
- Autumn 2023
- Publisher
- University of Iowa
- DOI
- 10.25820/etd.006847
- Number of pages
- xvi, 167 pages
- Copyright
- Copyright 2023 Madeline R Hines
- Grant note
- This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin (AR070914), other National Institutes of Health [R01 DK124510-01 and R01 HL153532-01A1], and the Department of Defense (W81XWH-15-0642).
- Language
- English
- Date submitted
- 12/04/2023
- Description illustrations
- illustrations, graphs
- Description bibliographic
- Includes bibliographical references.
- Public Abstract (ETD)
- Osteoarthritis is a degenerative joint disease involving the loss of cartilage. Prior research has considered oxidative stress, particularly when present as oxidation in cellular fat or fatty acids, as a catastrophic event leading to osteoarthritis. However, this thesis demonstrates that oxidation of fat normally considered oxidative stress is important to cartilage health and regulation of cellular metabolism. To investigate these processes, rigorous studies were done by modulating fat-based oxidative stress in healthy, injured, aged, and osteoarthritic cartilage. These studies demonstrate that fatty acid oxidation is important for regulating metabolism in response to tissue mechanical load and other stresses. This regulation appears to be protective against mechanical injury, by downregulating oxidative damage, suggesting fatty acid oxidation is a key regulator of injury response. Next, we investigated the effect of preventing fatty acid oxidation during aging. The results of this study demonstrated that prolonged exposure to a fatty acid antioxidant enzyme, glutathione peroxidase 4, leads to metabolic dysregulation. This indicates fatty acid oxidation is critical for cartilage cell metabolic regulation during aging. Finally, in an injury model of osteoarthritis, glutathione peroxidase 4 did not alter the outcome of end-stage osteoarthritis, in agreement with the clinical results from supplementing lipid antioxidants. From these studies we conclude that fatty acid oxidation regulates the cellular metabolism of chondrocytes thereby mediating the optimal health of cartilage.
- Academic Unit
- Free Radical and Radiation Biology Program
- Record Identifier
- 9984546848602771
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