Thesis Defence: Investigating the fibrotic effects of linoleic acid metabolites

Clara Letef, supervised by Dr. Sanjoy Ghosh, will defend their thesis titled “Investigating the fibrotic effects of linoleic acid metabolites” in partial fulfillment of the requirements for the degree of Master of Science in Biochemistry and Molecular Biology.

An abstract for Clara Letef’s thesis is included below.

Defences are open to all members of the campus community as well as the general public. Registration is not required for in person defences.

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ABSTRACT

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.