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Letef, Clara Anne

University of British Columbia

The North American diet is rich in omega-6 fatty acids, with linoleic acid (LA) being the most common. LA is blamed for heart muscle disease characterized by oxidative stress, fibrosis, and mitochondrial dysfunction. However, exact pathways of damage remain elusive. LA gets converted to oxidized linoleic acid metabolites (OXLAMs) by lipoxygenase (LOX). This thesis investigated how OXLAMs such as 13-hydroperoxyoctadecadienoic acid (13-HpODE), 13-hydroxyoctadecadienoic acid (13-HODE), 9-hydroperoxyoctadecadienoic acid (9-HpODE), and 9-hydroxyoctadecadenoic acid (9-HODE) impact mitochondrial redox status and fibrotic pathways using an in vitro model of NIH/3T3 fibroblasts. Cells were dosed with LA with or without 9c(i472) (an inhibitor of 15-LOX). Fibroblasts under LA showed decreased mitochondrial redox status with increased soluble nascent collagen as signs of stress. Using 9c(i472) restored mitochondrial redox potential and promoted deposited collagen instead. Inhibiting 13-HpODE production increased collagen III gene (the elastic subtype) and decreased collagen I gene representing the stiff collagen isoform. This indicates that LA’s bioconversion to 13-HpODE is critical for promoting ‘stiffness’ with LA treatment. To investigate the role of oxidative stress, H₂O₂ was added to fibroblasts treated with 9- or 13-HpODE. Although mitochondrial redox status dropped in both groups with H₂O₂, the drop was larger with 13-HpODE. This indicated a greater consumption of mitochondrial antioxidants by 13-HpODE than 9-HpODE. After its production, HpODEs is cleared to non-reactive HODEs using glutathione (GSH), as the co-factor. Supplementing fibroblasts with GSH could not reverse the drop in mitochondrial potential with H₂O₂ and HpODEs. However, adding GSH did improve the redox potential in LA treated cells. This indicates that LA through some unknown mechanism promotes greater utilization of GSH than its oxidized derivatives. Finally, gene expression of other mitochondrial antioxidants like catalase and SOD2 remained unchanged with both OXLAM treated fibroblasts, negating any potential major role. This thesis provided the framework on how 13-HpODE rather than its precursor LA, might be responsible for the fall of mitochondrial redox status and increased ‘stiff’ collagen production within fibroblasts, contributing to downstream fibrosis and heart failure.

Text

2024-04-29

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/88090

Biochemistry and Molecular Biology

University of British Columbia

UBCO

http://creativecommons.org/licenses/by-nc-nd/4.0/