Science, Irving K. Barber Faculty of (Okanagan)
DSpace
UBCO
Letef, Clara Anne
Master of Science - MSc
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.
INVESTIGATING THE FIBROTIC EFFECTS OF OXIDIZED LINOLEIC ACID METABOLITES by Clara Anne Letef B.Sc., The University of British Columbia, 2022 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE COLLEGE OF GRADUATE STUDIES (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) April 2024 © Clara Anne Letef, 2024 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis entitled: Investigating the fibrotic effects of oxidized linoleic acid metabolites submitted by Clara Anne Letef in partial fulfillment of the requirements of the degree of Master of Science . Dr. Sanjoy Ghosh, Irving K. Barber Faculty of Science Supervisor Dr. Kirk Bergstrom, Irving K. Barber Faculty of Science Supervisory Committee Member Dr. Isaac Li, Irving K. Barber Faculty of Science Supervisory Committee Member Dr. Ismail Laher, Professor, The University of British Columbia (Vancouver) University Examiner iii 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. iv Lay Summary The North American diet is filled with seed oils like sunflower oil that might be behind recent rises in heart disease rates across Canada. Diseased hearts show damage to parts of the cell called mitochondria and are full of hard tissue made of collagen. Previous research has linked excess omega-6 fats in seed oils to mitochondrial damage and excess collagen. Currently, the regulation of collagen production by omega-6 fats is unclear. As the rates of heart disease and seed oil consumption both continue to increase, identifying links between the two has become crucial for the health of Canadians. My thesis shows that rather than omega-6 fats per se, a specific metabolite of these omega-6 fats is responsible for this increase in collagen-induced stiffening and mitochondrial damage. This encourages new approaches where we can block production of these metabolites and continue to use vegetable oils while preventing heart disease among Canadians. v Preface The design of this Master’s research program belongs in part to Dr. Sanjoy Ghosh in collaboration with Clara Letef. All experiments and analyses were performed by Clara Letef. vi Table of Contents Abstract .......................................................................................................................................................iii Lay Summary .............................................................................................................................................. iv Preface .......................................................................................................................................................... v Table of Contents ........................................................................................................................................ vi List of Tables ............................................................................................................................................... ix List of Figures ............................................................................................................................................. x. List of Abbreviations ................................................................................................................................ xii. Acknowledgements ...................................................................................................................................xiii Dedication .................................................................................................................................................. xiv Chapter 1: Introduction .............................................................................................................................. 1 1.1 Our diet is SAD .......................................................................................................................... 1 1.1.1 Ultra-processed Seed Oils ............................................................................................... 2 1.2 Fatty Acids ................................................................................................................................. 3 1.2.1 Types Fatty Acids ................................................................................................................ 3 1.2.2 Omega-6 excess and chronic inflammation......................................................................... 5 1.2.3 Reactive Oxygen Species (ROS) ......................................................................................... 6 1.3 Inflammation and Oxidative Stress ................................................................................................. 8 1.3.1 Mitochondrial Dysfunction.................................................................................................. 8 1.3.2 Linoleic Acid and its Bioconversion ....................................................................................... 9 1.3.3 Oxidized Linoleic Acid Metabolites (OXLAMs) .............................................................. 11 1.3.4 9- and 13-HpODEs and HODEs ........................................................................................ 13 1.4 Cardiac Fibroblasts ....................................................................................................................... 15 1.4.1 Collagen ............................................................................................................................ 17 1.4.1.1 Collagen Production ................................................................................................ 17 1.4.1.2 Types of Collagens ................................................................................................... 18 vii 1.4.2 Fibrosis .............................................................................................................................. 18 1.4.3 Cardiac Fibrosis and Heart Failure .................................................................................... 19 1.5 Hypothesis and Aims ............................................................................................................... 20 Chapter 2: The fibrotic implications of oxidized linoleic acid metabolites .......................................... 21 2.1 Overview ....................................................................................................................................... 21 2.2 Materials and Methods .................................................................................................................. 21 2.2.1 In vitro cell model.............................................................................................................. 21 2.2.2 Reagent and Treatment Preparations ................................................................................. 22 2.2.3 MTT Assay ........................................................................................................................ 23 2.2.4 Picosirius Red Staining ...................................................................................................... 23 2.2.4.1 Soluble Collagen Assay ............................................................................................ 23 2.2.4.2 Deposited Collagen Assay ........................................................................................ 24 2.2.5 RNA extraction, cDNA synthesis, and qPCR ................................................................... 24 2.2.6 Superoxide Detection assay ............................................................................................... 26 2.2.7 Statistical Analysis ............................................................................................................ 27 2.3 Results ..................................................................................................................................... 27 2.3.1 Linoleic Acid reduces mitochondrial redox potential when compared to oleic acid. ........... 27 2.3.2 Linoleic acid increases soluble and deposited collagen production. ..................................... 28 2.3.3 Inhibiting 13-HpODE production at high LA doses restores mitochondrial redox potential and increases deposited collagen formation. .................................................................................. 30 2.3.4 Inhibiting 13-HpODE increases Collagen III and decreases Collagen I gene expression dose-dependently. ........................................................................................................................... 32 2.3.5 Oxidatively stressed fibroblasts treated with HpODEs significantly reduces redox potential which cannot be restored with GSH supplementation. ................................................................... 33 2.3.5.1 9- and 13-HODE bioconversion pathways may be governed by different antioxidant systems. ...................................................................................................................................... 35 viii 2.3.6 GSH supplementation restores mitochondrial redox potential with high doses of LA. ........ 38 2.4 Discussion ................................................................................................................................ 38 Chapter 3: Conclusion ............................................................................................................................... 44 3.1 Significance of Findings .......................................................................................................... 44 3.2 Limitations of research and Future work ................................................................................. 45 3.3 Main Conclusions..................................................................................................................... 45 References .................................................................................................................................................. 47 Appendices.................................................................................................................................................. 56 Appendix A ......................................................................................................................................... 56 Appendix B ......................................................................................................................................... 57 Appendix C ......................................................................................................................................... 58 ix List of Tables Table 2.1 qPCR Primer Sequences………………………………………………………………...26 x List of Figures Figure 1: Trends of Fat availability per capita from 1909-2009 in the United States. Figure from Lee et al., 2022 (Lee et al., 2022) ............................................................................................................................. 1 Figure 2: PUFAs control inflammation through the actions of cyclo- and lipoxygenases resulting in immunoneutral or pro-inflammatory mediators such as leukotrienes and prostanoids. ................................ 6 Figure 3: Bioconversion pathways of LA. Figure from Szczuko et al., 2020 ............................................. 10 Figure 4: OXLAM conversion pathways from parent metabolite, linoleic acid (Figure from Contreras et al.,2020) …………………………………………………………………………………………………..12 Figure 5: Glutathione recycling and use by glutathione peroxidases 1 and 4. Image from Stolwijk et al., 2020 ............................................................................................................................................................. 14 Figure 6: Summary of 9- and 13-OXLAM synthesis .................................................................................. 15 Figure 7: Fibroblast activation cycle. Image from Gibb et al., 2020 .......................................................... 17 Figure 8: MTT Assay of LA and OA treated fibroblasts ............................................................................ 28 Figure 9: Soluble Collagen Assay of LA or OA treated fibroblasts ............................................................ 29 Figure 10: Deposited Collagen Assay of LA or OA fibroblasts. ................................................................. 29 Figure 11: Sirius Red staining of deposited collagen in BSA or LA treated fibroblasts ............................. 30 Figure 12: MTT Assay of LA and 9c(i472) treated fibroblasts. .................................................................. 31 Figure 13: Deposited Collagen Assay of LA and 9c(i472) treated fibroblasts. ........................................... 31 Figure 14: Soluble Collagen Assay of LA and 9c(i472) treated fibroblasts ............................................... 32 Figure 15: Gene expression of Collagen III and Collagen I with or without 15-LOX inhibition ............... 33 Figure 16: MTT Assay of 9-or 13-HpODE ±2mM H2O2, ±2mM GSH, or both, H2O2 and GSH ............... 34 Figure 17: Soluble Collagen Assay of 9-or 13-HpODE ±2mM H2O2, ±2mM GSH, or both, H2O2 and GSH.. ........................................................................................................................................................... 35 Figure 18: Deposited Collagen Assay of 9-or 13-HpODE ±2mM H2O2, ±2mM GSH, or both, H2O2 and GSH.. ........................................................................................................................................................... 35 Figure 19: GPx1 and GPx4 gene expression of LA treated fibroblasts ± 9c(i472) and GSH ..................... 37 xi Figure 20: Relative fluorescence of SOD activity with HpODEs and HODEs. ......................................... 38. Figure 21: MTT Assay of LA and GSH fibroblasts ................................................................................... 38 xii List of Abbreviations 13-HODE 13-hydroxyoctadecadienoic acid 13-HpODE 13-hydroperoxyoctadecadienoic acid 9-HODE 9-hydroxyoctadecadienoic acid 9-HpODE 9-hydroperoxyoctadecadienoic acid ANOVA Analysis of variance ARA Arachadonic acid ATP Adenosine triphosphate cDNA Complementary DNA FA Fatty acid GPx1 Glutathione peroxidase-1 GPx4 Glutathione peroxidase-4 GSH Glutathione H2O2 Hydrogen Peroxide HCl Hydrochloric acid HpETE Hydroperoxyeicosatetraenoic acid LA Linoleic acid MTT 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide MUFA Monounsaturated fatty acid NaOH Sodium hydroxide OA Oleic acid oxo-ODE Oxo-octadecadienoic acid PBS Phosphate buffered saline PUFA Polyunsaturated fatty acid RNA Ribonucleic acid ROS Reactive oxygen species xiii Acknowledgements I would like to first thank my supervisor, Sanjoy Ghosh. Thank you for taking a chance on me and encouraging to pursue research. You opened my eyes to a different way of thinking, and I cannot thank you enough. Your passion and mentorship these past three years have shown me there is a lot more to life than research and academics, and that there is a way to enjoy both. I sincerely look forward to working with you in the future. Thank you to my committee members, Dr. Kirk Bergstrom and Dr. Isaac Li. Thank you Dr. Bergstrom for providing multiple opportunities for me to collaborate with the Bergstrom Lab and yourself on incredible research, and Dr. Li, for your valuable input and perspectives during our committee meetings. You have both shown me many ways to critically analyze my own work, and this is something I plan to take forward on my next pursuit. To my current lab members, Amanda and Trisheeta. I don’t know if I would’ve been able to finish this project without you, and I will forever cherish our times in Mexico. Thank you for every ounce of support and every laugh you both provided me. I want to thank Sandy and Melody for their endless patience with me while teaching me the ropes of the Ghosh lab. I want to also extend a thank you to my colleagues in ASC 365, you made this journey unforgettable. A huge thank you to Jacqueline Barnett and Jessica Josephson in the Gibson Lab for all of your help teaching me the lab and life skills I needed to succeed, and for always being there, ready to help. To every individual on the administrative side at the University of British Columbia Okanagan who I may have crossed paths with over email or in person, thank you for keeping things running smoothly and answering any questions I had. I want to extend a special thank you to Lindsey Odne and Dr. Trudy Kavanagh. Finally, to my friends and family, thank you for always being there for me, even if you didn’t know what was going on. I am forever grateful to have each of you in my life. xiv Dedication This thesis is dedicated to my family. My parents, Charbell and Grace Letef, my siblings, Laura and Christopher, and our dog, Wally. 1 Chapter 1: Introduction Hi 1.1 Our diet is SAD Considering the Standard American Diet can be abbreviated as SAD, it should not come as a surprise that our population’s health has been declining (Carrera-Bastos et al., 2011), (Willett et al., 2015). Rich in processed foods, refined carbohydrates, and high fat dairy and meat products, this diet is the most popular amongst North Americans, and commonly referred to as the “Western diet” (Rakhra et al., 2020). Rising prices of whole foods over last decades has left people struggling to make ends meet with no choice to but to choose the “cheaper” option, with those often being pre-packaged processed foods (Rochefort et al., 2022). Prior to the 1960s, butter, lard, and plant-based saturated fats were the most popular dietary sources of fat (Lee et al., 2022). The widespread industrial use of vegetable oil began in 1965 (Lee et al., 2022). The tasteless, odorless, and high-smoke point qualities of vegetable oils made them seemingly great alternatives to saturated fat-filled fats such as butter, or lard, or even monounsaturated fat-filled olive oil (Thomas, 2000). Since their introduction in Canada, the vegetable oil market has been an integral component of the Canadian economy, holding large shares in exports (Agriculture Canada, 2018). Figure 1: Trends of Fat availability per capita from 1909-2009 in the United States. Figure from Lee et al., 2022 (Lee et al., 2022). Image used with permission under CC BY. 2 While the market and economy benefitted from the growing rates of vegetable oil production and consumption, recent decades have also seen growing rates of obesity, diabetes, heart disease and other non-communicable disease (NCDs) (Lee et al., 2022). While many theories have been proposed on the origins of this surge (e.g. high refined sugars or lack of exercise), the addition of vegetable /seed oils (such as corn, sunflower or soybean oil), is temporally related to the rise of these diseases, posing the question of causation or correlation between these two factors (Lee et al., 2022). 1.1.1 Ultra-processed Seed Oils While most people do try and pick healthier options, the addition of ultra-processed seed oils to commonly marketed foods is mostly beyond the average consumer’s control. The most common seed oils in Canada include corn, canola, and sunflower oil (Agriculture Canada, 2018). These oils are present in many processed and packaged foods and are grouped as vegetable oils (Thomas, 2000). The increase in vegetable oil consumption has been predicted to be following an upwards trajectory with Statista reporting 94.9 million metric tons increasing to 96 million metric tons from 2020 to 2021 alone (Statista, 2024). They also report that the edible oil market (vegetable oils and olive oil) is expected to grow up to 6.1% by 2025, once again prompting further investigation of how this volume of oil consumed will impact physiological functioning (Statista, 2024). While there are still large knowledge gaps throughout this body of literature, the currently consumed concentrations of vegetable oils, richest in omega-6 polyunsaturated fatty acids (PUFAs), have been linked to pro-inflammatory markers of NCDs (Banshi Saboo et al., 2019), (Mannuccio Mannucci et al., 2023). Following the (poorly) recommended switching of saturated fatty acids to PUFAs in the 1960s, the North American population was left consuming omega-6 PUFAs at a disproportional rate. While replacing SFAs with PUFAs reduced total blood cholesterol, multiple studies fail to show any cardiovascular benefits from this switch (Hamley, 2017), (Sacks et al., 2017). Subsequent studies showed that while excess omega-6 PUFAs cause detrimental cardiovascular health, omega-3 PUFAs from fish oils are cardioprotective (Harris et al., 2009), (Oppedisano et al., 2021). These two outcomes have been 3 closely linked to diseases such as cardiac fibrosis, a major contributing factor to most heart diseases (Harris et al., 2009), (DiNicolantonio & O’Keefe, 2018). 1.2 Fatty Acids 1.2.1 Types Fatty Acids Fatty acids are composed of a hydrophilic carboxylic head and a hydrophobic hydrocarbon tail, making them amphipathic molecules that make up every cell membrane, most hormones, and other cell-mediating secondary messengers in the body (de Carvaldo and Caramujo, 2018), (Moss et al., 1995). Depending on the properties of their hydrocarbon tails, such as the number of carbons, hydrogens, and branching patterns, they can typically be divided into three groups: saturated (SFA), mono-unsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs) (Moss et al., 1995). SFAs lack double bonds (that causes branching) and are linear in structure. In excess, this can allow for plaque formation in arteries due to “stacking” of the FAs (Sudheendran et al., 2010). A study of Canadian adults reported that approximately 10% of total daily energy is from SFA intake (Ahmed et al., 2021). The most common SFA’s in the Canadian diet include palmitic and stearic acid. denoted as C16:0, and C18:0 respectively. These FAs are obtained from foods such as butter, meat, and coconut oil, and are necessary for hormone synthesis, maintaining cell membrane integrity, and lipogenesis (van Rooijen and Mensink, 2020). Palmitic acid is typically consumed at a higher concentration due to its presence in meat and dairy products (Carta et al., 2017). MUFAs contain one double bond causing a slight bend in their hydrocarbon tail. The most common MUFA is oleic acid (OA) (C18:1n9) with its double bond located on the ninth carbon of 18, making it an omega-9 FA (Thomas, 2000). It can be synthesized in the body using the enzyme stearoyl-CoA desaturase, which adds a double bond to stearic acid (Piccinin et al., 2019). OA is therefore considered non-essential as it can be synthesized but it is also the most common MUFA consumed in the diet and comes from sources such as olive oil (Piccinin et al., 2019). MUFAs are typically used as experimental controls when investigating PUFAs as the minimal oxidation or bioconversion that occurs 4 with the one double bond in comparison to the two or more found in PUFAs. In this thesis, for in vitro experiments, OA was used as a fatty acid control for LA treated cells having the same carbon chain length but different number of double bonds. PUFAs are fatty acids that contain at least two double bonds in their hydrocarbon tails (Thomas, 2000). Essential PUFAs are usually classified as either omega-3 (n-3) or omega-6 (n-6) fatty acids, depending on the location of the first double bond from the terminal methyl group. Although not synthesized by animals, PUFAs are necessary to carry out functions such as maintaining brain health and mediating the inflammatory response (Bazinet and Layé, 2014). Omega-3s most famously come from foods such as fish, which contain docosahexanoic acid (DHA) and eicosapentaenoic acid (EPA) (Gil and Gil, 2015). The parent omega-3 PUFA, alpha linolenic acid (ALA) gets elongated (ELOVL1) then desaturated (FADS1) to become EPA, then elongated once more by ELOVL2 to become DHA ( Eltweri et al., 2017). DHA is an essential molecule for brain growth and development (Bazinet and Layé, 2014). On the other hand, omega-6 fatty acids contain the first double bond on the sixth carbon from the methyl terminus. The most common omega-6 PUFA in the Canadian diet is linoleic acid (LA), which is essential and comes from foods such as vegetable oils, meat, and eggs (Whelan and Fritsche, 2013). It can be converted through a series of enzymes to arachidonic acid (ARA) or be oxidized to metabolites such as HODEs (Whelan and Fritsche, 2013), prostaglandins (PGs) and leukotrienes (LTs), synthesized by cyclogeneses (COX) and lipoxygenases (LOX). ARA participates in the production of anti- and pro-inflammatory molecules and also constitute a large component of cell membranes (Tallima and El Ridi, 2018). HODEs are activators of the peroxisome proliferator-activated receptor (PPAR) family and are also markers of oxidative stress in diseases such as Alzheimer’s and atherosclerosis (Bojic et al., 2015), (Shinto et al., 2022). Prior to vegetable oils infiltrating food systems in the 1950s, North Americans consumed about one gram of omega-3 fat for every four grams of omega-6 (Simopoulos, 2011). The addition of seed oils in the mid-60s has left this population eating upwards of 20-25 times the amount of omega-6, and not 5 nearly enough omega-3 fats to compensate for the pro-inflammatory impact of this volume of omea-6 consumed (Patterson et al., 2012), (Simopoulos, 2011). A study focusing on subcutaneous adipose tissue presents a staggering 9.1% to 21.5% increase in LA from 1959-2008 (Guyenet and Carlson, 2015). Statistics Canada reported a 54% increase in the amount of PUFAs consumed by Canadians from 1976 to 2005 (Harrison et al., 2019). The serious increase in n-6 consumption (specifically with the increase of LA concentration in the diet) but not n-3 consumption has repeatedly been linked to chronic diseases (Simopoulos, 2011). By investigating LA’s bioconversion to its downstream metabolites, a deeper understanding of how these diseases continue to rise at a determinantal rate in our population could lead to potential cures through lifestyle changes and novel treatments. 1.2.2 Omega-6 excess and chronic inflammation Chronic inflammation is categorized as long-term low grade systemic inflammation (Bo et al., 2021). It often begins when an acute inflammatory stressor is not adequately eradicated or when there is prolonged oxidative stress that results in mitochondrial dysfunction (Pahwa et al., 2023). This persistent, low-grade inflamed state leaves the body unable to fully restore antioxidant and immune defenses to combat future stressors, and keeps the body in a “pro-inflammatory” state of responding to stimuli (Boutens and Stienstra, 2016), (Wensveen et al., 2015). Chronic inflammation has been long linked to chronic inflammatory diseases such as cardiovascular disease, diabetes, and obesity, all three of which are prevalent at overwhelming rates in the Canadian populations (Lee et al., 2022), (Public, 2017). Over the past few decades, these NCDs have been investigated from the dietary angle, primarily focusing on fats (Patterson et al., 2012). As discussed, the link between excess omega-6 PUFAs and NCDs may come from the high concentrations of LA in the diet and the downstream metabolites (primarily, arachidonic acid) being linked to pro-inflammatory prostaglandins, leukotrienes, and thromboxanes (Figure 2) (Heller et al., 2003). PUFAs control inflammation in the body and if there is substrate (LA), the downstream inflammatory markers will continue to be made and act on cells and tissues. With these rising rates of vegetable oil usage across Canada, a food industry where omega-6 fats and LA are not provided and consumed in excess has become increasingly less likely. 6 Figure 2: PUFAs control inflammation through the actions of cyclo- and lipoxygenases resulting in immunoneutral or pro-inflammatory mediators such as leukotrienes and prostanoids. Figure adapted from Heller et al., 2003 (Heller et al., 2003). SFA; saturated fatty acid, MUFA; monounsaturated fatty acid, PUFA; polyunsaturated fatty acid, OA; oleic acid, EPA; eicosapentaenoic acid, AA; arachidonic acid, PLA2; phospholipid lipase 2, TXA; thromboxane, PG; prostaglandin, PGI3; prostacyclin inhibitor, LT; leukotriene, LTC; leukotriene, LTE; leukotriene. 1.2.3 Reactive Oxygen Species (ROS) To preface this introduction to reactive oxygen species (ROS), it should be noted that the body and every cell has been designed to combat these and that the chronic exposure to ROS or improperly functioning neutralization responses can become problematic (Ho et al., 1997). ROS can be beneficial or harmful, with many critical signaling pathways needing to be regulated by ROS to maintain cellular homeostasis (Ray et al., 2012). It is the dysregulation or inability for the cell to combat excessive ROS stressors that contributes to disease formation. Most diseases are associated with an accumulation of ROS, as has been suggested for cancer, Alzheimer’s disease, diabetes, cardiovascular disease (CVD), inflammatory bowel disease (IBD), and non-alcoholic fatty liver disease (NAFLD) (Alfadda and Sallam, 2012). The endoplasmic reticulum and mitochondria are responsible for neutralization of ROS (Alfadda and Sallam, 2012). Most ROS are highly reactive molecules that arise from the mitochondria’s electron transport chain (ETC). Mitochondria deploy multiple mechanisms to avoid oxidative damage, including systems such as glutathione peroxidases, catalases, or superoxide dismutase (Alfadda and Sallam, 2012). 7 There are eight types of glutathione peroxidases (abbreviated as GPx1-GPx8). These enzymes have an essential cofactor of glutathione (GSH) and are a vital step in glutathione recycling as seen in Figure 5. In terms of fatty acid oxidation and mitochondrial protection in relation to this thesis, GPx1 and GPx4 will be further investigated. Both of these isoforms (as well as GPx2 and GPx3) are selenoproteins containing selenocysteine (Brigelius-Flohé & Maiorino, 2013). GPx1 is localized to the cytoplasm and mitochondria, expressed throughout cell types, and is used to neutralize hydrogen peroxides (H2O2) to produce water and oxidized glutathione (GSSG) (Brigelius-Flohé and Maiorino, 2013). Although the most commonly used GPx, GPx1 knock-out mice are not be at any physiological disadvantage when compared to the wild-type, and develop normally (Ho et al., 1997). GPx4, also referred to as phospholipid hydroperoxide glutathione peroxidase, has three isoforms and locations within the cell (Brigelius-Flohé and Maiorino, 2013). Cytosolic (cGPx4), mitochondrial (mGPx4), and sperm nuclear (snGPx4) (Brigelius-Flohé and Maiorino, 2013). cGPx4 knockout is lethal and is considered essential to life (Seiler et al., 2008). GPx4 is the only GPx isoform capable of reducing lipid hydroperoxides present in the cell membrane, including PUFAs (Seiler et al., 2008). These PUFAs are converted through 12 or 15-lipoxygenase and the product (HpODE) acts as the ideal substrate for GPx4. GPx4 is able to reduce this lipid peroxide and prevent buildup within the cell, which this thesis sets out to show could influence mitochondrial dysfunction and downstream physiological impacts. The second primary antioxidant system, superoxide dismutase (SOD) is involved in superoxide (O2•– ) ion neutralization to H2O2 (Hayyan et al., 2016). Compared to catalase and glutathione peroxidases which convert H2O2 to water (catalase and GPx1) or GPx4’s function in neutralizing lipid peroxides. SOD has three isoforms, one in soluble form (SOD1), one in the mitochondria (SOD2), and one present extracellularly (SOD3) (Litwack, 2018). The superoxide ion is created through the ETC in the mitochondria, and NADH oxidase, being a product of respiration and making it critical to immune regulation (Hayyan et al., 2016). Clearly essential to overall cell functioning, these three antioxidant systems work together to maintain mitochondrial health. 8 While the mitochondria are equipped with these defenses, dietary intake does play a role in molecular-level health. There have been countless studies showing that excess n-6 consumption leads to chronic inflammation through mitochondrial dysfunction and that switching to diet plans such as a Mediterranean diet- that is rich in fruits, nuts, fish, and leafy vegetables- could potentially improve these effects (Khalil et al., 2022). This study together with multiple others have shown decreased SOD, GPx, and catalase activity when it comes to a diet high in PUFAs (Diniz et al., 2004), (Khalil et al., 2022) This thesis focused on building upon these conclusions, further investigating how the reactive intermediates (HpODEs) in LA bioconversion, the most common dietary n-6 PUFA, play a role in inducing mitochondrial dysfunction and what that means in terms of cardiovascular health in vitro. 1.3 Inflammation and Oxidative Stress 1.3.1 Mitochondrial Dysfunction Mitochondria are well known as the powerhouses of the cell. This tiny organelle is arguably the most important part of the cell. It produces all the energy needed for cellular processing, signaling, and provides antioxidant responses needed to eliminate oxidants and other stressors of the cell (Apostolova & Victor, 2015). Without the mitochondria, a cell is unable to perform its function. For example, if fibroblasts experience severe acute oxidative stress, or chronic reactive oxygen species (ROS) exposure, the mitochondria are left focusing the cell’s energy reserves in combatting these threats, rather than providing ample energy for the cell to perform its function, in this case, producing collagen (Siwik et al., 2001). One of the causes of chronic inflammation is mitochondrial dysfunction due to inflammatory markers or prolonged ROS exposure (Pahwa et al., 2023). Much like chronic inflammation, many diseases can be traced back to mitochondrial dysfunction, including cardiovascular disease, fibrosis resulting in CVD or fatty liver disease. Mitochondrial redox potential can be used to indicate mitochondrial dysfunction and oxidative stress within the cell, as well as its health and functioning (Handy & Loscalzo, 2012). Redox potential within the mitochondria can be determined by the balance of oxidized and reduced forms of molecules and it has been shown that a dysregulation in either oxidized or reduced products can indicate the cell is 9 undergoing apoptosis or another change in function, such as the conversion of fibroblasts to pro-fibrotic myofibroblasts in NIH3T3 cells (Kang & Pervaiz, 2012) (Negmadjanov et al., 2015). Assays such as the MTT assay can measure the efficiency of succinate dehydrogenase (within the electron transport chain) to approximate the health of mitochondria and its ability to produce energy for the cell (Mosmann, 1983). With this said, mitochondria are heavily equipped with mechanisms to avoid immediate dysfunction, including various antioxidant systems, as well as redundancy in mitochondrial pathways in which multiple systems must be depleted before the mitochondria stop functioning. When oxidative stress is experienced by the mitochondria (whether it be from chronic inflammatory markers, or processes such as aging), there is always potential for dysfunction (Amorim et al., 2022). Although these mechanisms may be able to prevent long-term damage, in situations where a stressor is not removed, specifically in terms of dietary intake, there is no “letting up” of this stimulus and can result in cellular death (Juszczyk et al., 2021). When there is enough cell death, advancements in disease progression in terms of physiological changes can start to be seen. This brings us to a large gap in fatty acid literature, with the link between LA bioconversion and downstream physiological results of mitochondrial dysfunction. This thesis focuses on how the oxidized products of LA may impact mitochondrial health, and in turn influence fibrosis development in the heart, using in vitro models of fibroblasts. There have already been studies suggesting that 13-HpODE and 13-HODE accumulation can cause mitochondrial degradation (van Leyen et al., 1998), (Kühn and Brash, 2020). While 13-HODE has been denoted an “anti-inflammatory” molecule, these findings prompted further investigations of reactive intermediates of this pathway (HpODEs). 1.3.2 Linoleic Acid and its Bioconversion The most famous n-6 PUFA, linoleic acid, is an 18-carbon fatty acid with two double bonds, denoted as C18:2n-6 by IUPAC (PubChem, 2024). Of the n-6 PUFAs present in seed oils, LA is of the highest concentration (Poli et al., 2023). As it is the parent molecule, LA can either be converted to ARA (through desaturases), or to oxidized LA metabolites (OXLAMs), such as HODEs (Szczuko et al., 2020). 10 While it is unknown whether ARA or OXLAMs are the preferential bioconversion pathway for LA, current diet trends and absurdly high concentrations of LA in the diet, has left more than enough for either pathway to be utilized by the parent molecule (Poli et al., 2023). Figure 3: Bioconversion pathways of LA. Figure from Szczuko et al., 2020. HODE; hydroxyoctadecadienoic acid, oxo-ODES; oxooctadecadienoic acids, HpETEs; hydroperoxyeicosatetraenoic acid, HETE; hydroxyeicosatetraenoic acid, LOX; lipoxygenase, COX; cyclooxygenase, EEE; epoxyeicosatrienoic acid. CYP; cytochrome p450 enzyme. Image used with permission under CC BY. As briefly mentioned above, PUFAs also act as substrates for cyclooxygenases (COX) and lipoxygenases (LOX), synthesizing downstream immunoneutral or pro-inflammatory leukotrienes, prostanoids, and prostaglandins (Heller et al., 2003). Omega-3 PUFAs, such as EPA, are converted through COX and LOX enzymes to become immunoneutral prostanoids or leukotrienes, whereas omega-6 PUFAs, such as ARA undergo the same process to become pro-inflammatory molecules (Heller et al., 2003). ARA undergoes conversion to downstream metabolites through three main enzyme families, LOX, COX, and cytochrome P450 (CYP) (Tallima & El Ridi, 2018). LOX enzymes are responsible for the production of hydroxyeicosatetraenoic acid (HETEs), oxoeitosatetraenoic acids (oxo-ETEs), and lipoxin A4 (Powell & Rokach, 2015). HETEs and oxo-ETEs have been seen as by-products of thromboxane and Linoleic Acid (LA) γ-linoleic acid (GLA) dihomo-γ-linoleic acid (DGLA) 11 leukotriene synthesis but have also (in high concentrations) been found to be PPAR family regulators, immune mediators, as well as markers in progressed forms of diabetes and asthma (Weaver et al., 2012) (Muro et al., 2003). CYP enzymes synthesize HETEs as well as epoxyeicosatrienoic (EETs) which have been shown to induce cancer tumour growth (Rand et al., 2017). ARA also undergoes conversion through the COX enzyme pathway, producing pro-inflammatory thromboxanes and prostaglandins, both of which have been linked with cancer and tumourogenesis, as well as pain mediation (Wang & DuBois, 2010) (Smith et al., 1998). 1.3.3 Oxidized Linoleic Acid Metabolites (OXLAMs) One of the main bioconversion pathways of LA produces oxidized linoleic acid metabolites (OXLAMs). OXLAMs are a family of metabolites classified as bioactive metabolites of LA and are converted by lipoxygenases (Fang et al., 1999). They are heavily involved in pain mediation and have also been found involved in neuron activation (Patwardhan et al., 2009). Of the main OXLAMs, 9- and 13-hydroxyoctadecadienoic acid (9-HODE and 13-HODE) are the most abundant in humans (Vangaveti et al., 2010). As seen in Figure 4, non-enzymatic (NE) conversions and lipoxygenases (LOX) are responsible for the first oxidation of LA to hydroperoxyoctadecadienoic acids (HpODEs). Although HpODEs are short lived, they are reactive intermediates that have been shown to be involved as PPARγ family regulators, as well as oxidative glutathione inducers (Snodgrass and Brüne, 2019), (Nagy et al. 1998). HpODEs are then converted by the glutathione peroxidase family of enzymes (usually, phospholipid hydroperoxide glutathione peroxidase, or GPx4) which is present on the mitochondrial membrane to become stable inflammatory mediators, hydroxyoctadecadienoic acids, known as HODEs (Murphy and Zarini, 2002). HODEs are then converted to oxo-octadecadienoic acids (oxoODEs), which are short-lived oxidized OXLAMs (Hamblin et al., 2009). 12 Figure 4: OXLAM conversion pathways from parent metabolite, linoleic acid. (Figure from Contreras et al., 2020). NE; non-enzymatic pathway, LOX; lipoxygenase, GPx; glutathione peroxidase enzyme, hydroxy FADH; hydroxy fatty acid dehydrogenase, HPODE; hydroperoxyoctadecadienoic acid, HODE; hydroxyoxtadecadienoic acid, oxo-ODE; oxo-octadecadienoic acid. Image used with permission under CC BY. 9-HODE has been categorized as a “pro-inflammatory” mediator of inflammation while 13-HODE has been denoted “anti-inflammatory” (Vangaveti et al., 2010). Unfortunately, the sheer volume of conflicting literature has made it difficult for any real conclusions to be made for OXLAMs. 13-HODE specifically has been said to have protective effects in early atherosclerosis but then contributed to apoptosis and plaque formation later in the disease (Szczuko et al., 2020) (Hampel et al., 2006). 13-HODE has also been denoted a transient receptor potential vanilloid-1 (TRPV1) agonist leading to inflammatory hyperalgesia (Patwardhan et al., 2009) but at the same time, as an endogenous ligand that activates PPARγ and downregulates PPARβ to overall decreases the inflammatory response (Hamblin et al., 2021). From these three statements alone, there can be no conclusion made on the effects of 13-HODE nor could it be classified as a pro or anti-inflammatory metabolite. The same applies for 9-HODE. 9-HODE was also found to activate PPARγ (anti-inflammatory) but also contribute to macrophage regulation and release of interleukin-β1 (pro-inflammatory) (Nagy et al., 1998) (Hamblin et al., 2021) (Ku et alk., 1992). In 2020, Contreras et al. (2020) stated that among OXLAMs, 9-, 10-, and 12- HODEs are associated with pro-inflammatory responses, whereas 13-HODE, 9- and 13-oxoODEs can help resolve inflammation (Contreras et al., 2020). This large yet confusing body of literature surrounding OXLAMs and their conversion beginning with the excessive influx of dietary LA, sparked our interest in further 13 investigating the impacts of LA’s bioconversion to OXLAMs may have on mitochondrial health and physiological processes such as cardiac fibrosis. 1.3.4 9- and 13-HpODEs and HODEs This thesis will focus primarily on the bioconversion of LA to 9- and 13- labelled OXLAMs, excluding oxoODEs. 9-HpODE is formed through non-enzymatic (NE) pathways or 5-LOX, whereas 15-LOX1 converts LA to 13-HpODE (Snodgrass and Brüne, 2019). This enzyme primarily converts ARA to into 15-HpETE but prefers LA as a substrate (Snodgrass and Brüne, 2019). The other isoform, ALOX-15B, is not involved in the conversion of LA to 13-HpODE. 13(S)-HODE is preferentially synthesized over 9(S)-HODE and the ratio of 9- to 13-HODE is essential for proper inflammatory induction and resolution (Vangaveti et al., 2010). The influx and accumulation of LA and its conversion to OXLAMs can evidently present a problem with this ratio and may lead to chronic inflammation (Vangaveti et al., 2010). Figure 6 displays the conversion of LA to 9 or 13-OXLAMs. LA is oxidized by 15-lipoxygenase to become 13-HpODE, an unstable reactive intermediate, capable of causing of causing oxidative stress, due to its double bonded oxygens. 9-HpODE is made through the conversion of LA through 5-lipoxygenase or non-enzymatic pathways (Contreras et al., 2020). There is limited research on the functions of HpODEs, relative to HODEs. On the mitochondrial membrane, glutathione peroxidase-4 (GPx4) oxidizes its essential cofactor, glutathione (GSH), to reduce HpODEs to HODEs, the more stable and abundant OXLAMs in the body. GSH is used up in this reaction and may not necessarily be recycled fast enough to compensate for concentration of substrate entering the GPx4 system (Wu et al., 2004) (Figure 5). Since these studies were completed to investigate the impacts of OXLAMs in terms of cardiac fibrosis, experiments supplementing glutathione to restore any depleted antioxidant reserves were made up to 2mM, the normal, healthy, physiological concentration (Aoyama, 2021). 14 Figure 5: Glutathione recycling and use by glutathione peroxidases 1 and 4. Image from Stolwijk et al., 2020. G6P; glucose-6-phosphate, G6PD; glucose-6-phosphate dehydrogenase, GR- glutathione reductase, GPx; glutathione peroxidase enzyme. Image used with permission under CC BY-NC-ND. HODEs are then converted by hydroxy fatty acid dehydrogenase (hydroxy FADH) to become ketones referred to as oxo-ODEs (Yuan et al., 2012). 9- and 13-oxoODEs have both been labelled PPARγ ligands, aiding in the anti-inflammatory response (Contreras et al., 2020). 13-oxoODE has also been known to react with GSH via GSH-transferase and be rapidly exported from the cell (Murphy and Zarini, 2002). GSH bound 13-oxoODE is unknown to have any biological effects and is currently considered to be exported from the cell to avoid 13-oxoODE accumulation in the mitochondria (Murphy and Zarini, 2002). 15 Figure 6: Summary of 9- and 13-OXLAM synthesis. Linoleic acid is converted to hydrooxyoctadecadienoic acids (9; and 13-HpODE) by non enzymatic (NE) or lipoxygenase activity (LOX). HpODEs are reduced [R] by glutathione peroxidase enzymes (GPx4) with essential cofactorglutathione (GSH) to hydroxyoctadecadienoic acid (HODEs). HODEs are oxidized [O] by hydroxy fatty acid dehydrogenase (hydroxy FADH) to oxo-oxtadecadienoic acids (oxo-ODEs) 1.4 Cardiac Fibroblasts The heart is made up of various cell types, including cardiomyocytes (electrical signaling and contractile forces), macrophages (immune and signal transduction), and fibroblasts (ECM maintenance, tensile strength, elastic strength, and cytokine secretion) (Ding et al., 2022). In the average heart, fibroblasts make up ~40% of cardiac cells and are essential for producing and maintaining collagen ratios in the heart (Ding et al., 2022). Dysregulation of this cell type can result in problems such as improper immune signaling, incorrect electrical conduction, as well as activation of a fibrotic phenotype which can lead to cardiac fibrosis (Souders et al., 2009). 16 Fibroblasts are a cell type whose primary function is to produce collagen and help lay down and maintain the framework of the extracellular matrix (ECM). Other than the heart, fibroblasts are present throughout the body and are a critical cell type involved in tissue and wound repair, as well as in the secretion of cytokines, contributing to immune regulation and (de)/activation of macrophages, for example (Plikus et al., 2021). In vitro studies with fibroblasts have been used to determine mechanistic links involving diseases such as fibrosis, as they can take a pro or anti-fibrotic phenotype (Souders et al., 2009). Fibroblasts produce ROS as a byproduct of cellular functioning. When fibroblasts are stimulated to produce collagen, the mitochondria must utilize the electron transport chain to produce enough ATP for this process, producing ROS as a byproduct of cellular respiration (Richter & Kietzmann, 2016). When fibroblasts are excessively stressed, for example, activated and chronically stimulated to produce excess collagen, there is excess ROS produced. In turn, this ROS can stimulate fibroblast proliferation and fibroblast activation, and therefore increase the amount of deposited collagen is being produced, contributing to fibrosis formation (Wang et al., 2023). Cardiac fibroblasts are responsible for secreting signals such as growth factor (GF), interleukin-1β (ILB1), interleukin-6, and tumour necrosis factor-α (TNFα) (Plikus et al., 2021). These are just some of the signals involved as matrix metalloproteases will respond to these pro- or anti-inflammatory signals and begin the process of cardiac remodelling (Sayer and Bhat, 2014). The other systems heavily involved in cardiac remodeling are the renin-angiotensin-aldosterone system and the sympathetic nervous system (Azevedo et al., 2016), (Sayer and Bhat, 2014). Whether from secreted signals, oxidative stress and mitochondrial dysfunction, activated angiotensin-II (AngII) and/or tumour growth factor-β (TGFβ) a pro-fibrotic phenotype is promoted amongst differentiated myofibroblasts, immune, and vascular cardiac cells activating excess collagen production and positive-feedback loops for the above profibrotic mediators, contributing to resistance in cell death and dysregulation in fibrotic homeostasis (Dai et al., 2011), (Gibb et al., 2020), (Saadat et al., 2021). A pro-fibrotic phenotype may be beneficial in the context of wound repair, but with prolonged secretion of activated AngII and other pro-inflammatory signals, such as 17 TGFβ, will leave cardiac fibroblasts in a chronic pro-inflammatory state, overproducing collagen, and contributing to pathologies such as fibrosis in the heart, one of the major drivers of cardiovascular disease (Gibb et al., 2020). Figure 7 shows the summary of the fibroblast activation cycle from Gibb et al., 2020 (Gibb et al., 2020). Figure 7: Fibroblast activation cycle. Image from Gibb et al., 2020. ECM: extracellular matrix, FN: fibronectin, AngII: Angiotensin II, TGFβ: transforming growth factor-beta, POSTN: periostin. Image used with permission. 1.4.1 Collagen 1.4.1.1 Collagen Production Collagen is an extracellular matrix structural protein secreted by fibroblasts throughout the body (Plikus et al., 2021). Collagen production begins with a chemical stimulus (likely TGFβ or AngII) or mechanical stimuli (wounds) activating gene expression. Ribosomes then synthesize pre procollagen chains which consists of three alpha chains which get modified by the Golgi apparatus to become procollagen (Canty and Kadler, 2005). This procollagen is considered “soluble collagen” and can be used as an indicator for initial stressors in the cell. This is due to oxidative or mitochondrial stress in fibroblasts that may cause them to prematurely release soluble forms of collagen into the ECM, changing the levels 18 and composition of soluble collagen (Ito and Nagata, 2021), (Ricard-Blum, 2010), (Plikus et al., 2021). After modification through the actions of prolyl hydroxylase and lysyl hydroxylase in the endoplasmic reticulum to become tropocollagen, a molecule of three procollagen chains (Canty and Kadler, 2005). Tropocollagen is then transported to the ECM where MMPs cleave tropocollagen to become collagen fibrils (Lu et al., 2011). Collagen fibrils are then acted upon by lysyl oxidase (LOX) performing a “cross-linking” step of fibrils that results in stable and insoluble collagen fibers in the ECM that can be used for structural and framework purposes (Lu et al., 2011). Certain collagen types (usually categorized by their location, function in the organism, and the procollagen gene expressed) undergo various modifications in the formation of tropocollagen step (Ricard-Blum, 2010). 1.4.1.2 Types of Collagens There are 4 major types of structural collagen (Type I-IV). Fibroblasts are responsible for secreting Type I and III collagen fibers (Chapman et al., 2016). Type I collagen functions to resist forces, tension, and stretch, and is one of the tougher types of collagens (Chapman et al., 2016). Type III collagen, often co-distributed with Type I, does the opposite. Type III provides structural support through elasticity and flexibility, allowing movement in certain organs, such as the heart beating (Chapman et al., 2016). Both I and III are involved in wound repair. Type I typically lays the framework in early wound healing, whereas Type III takes over in rapid growth and development, producing new tissue (Singh et al., 2023). The ratio of Type I and III collagen is essential throughout the body and dysregulation of this ratio can lead to diseases such as fibrosis (Singh et al., 2023). 1.4.2 Fibrosis Fibrosis can be a good thing. When we have a cut, fibrosis is what stops us from bleeding out, or getting a wound infected. In the context of wound repair, pro-fibrotic measures are something we want. When there is no wound but a pro-fibrotic phenotype amongst fibroblasts is present; we are presented with a problem. Excess production of collagen or a decrease in collagen degradation can lead to unwanted fibrosis, which can be a huge problem for certain organs such as the heart which must maintain stable ratios of collagen to other ECM components to properly pump blood (Singh et al., 2023). Another threat 19 that may lead to unnecessary fibrosis as mentioned in Sections 1.2.2 and 1.4 is chronic inflammation. Chronic inflammation can also stimulate the ECM to accumulate its collagen, which can form fibrotic scars, making it even harder for organs such as the lungs or heart to perform their functions of expansion and pumping, respectively (Wynn, 2007). 1.4.3 Cardiac Fibrosis and Heart Failure As described above, fibrosis can quickly become more detrimental than beneficial to an organ. Cardiac fibrosis has been heavily researched, with multiple contributors known today. The first being hormonal. Angiotensin II is part of the renin-angiotensin-aldosterone system and stimulates TGFβ1 which in turn, stimulates quiescent fibroblasts to become collagen secreting myofibroblasts. With continued stressors (such as oxidative stress or lack of downregulation in AngII signalling), these cells can differentiate into myofibroblasts and continuously secrete collagen which can cause disproportionalities in the makeup of the ECM (Rosenkranz, 2004). AngII is also a potent inducer of IL1β, a pro-inflammatory cytokine which plays the role of maintaining inflammation in the failing heart (Liu et al., 2014), (Long, 2001). In a study done by Beam et al., (2015) TGFβ1 signalling was shown to be induced by dietary intake of LA, manipulating stimulating lipoxygenase (LOX) as well as Type I collagen (Beam et al., 2015). Mitochondrial defects have also been linked to heart failure under high n-6 PUFA intake, when compared to high SFA intake (Galvao et al., 2011). The ratio of Type I and III collagen has also been shown to be a major contributor to “stiffening” of the heart when it comes to cardiac fibrosis (Beam et al., 2015). This study supplemented dietary LA in mice which resulted in increased collagen I, and decreased collagen III, causing stiffer heart tissue and abnormal cardiac blood flow greater than in their olive oil-treated counterparts (Beam et al., 2015). This abnormal flow is indicative of cardiac dysfunction (Beam et al., 2015). With increased collagen I production, myocardial tissue are of “stiff” phenotype, whereas increased collagen III would adopt an elastic phenotype (Collier et al., 2012). This link between collagen phenotype to downstream heart failure, a disease in which 1 in 3 Canadians will develop, was enough to prompt further investigation in 20 this fibrotic mechanism and its influence with increasing dietary intake of LA (Canadian Cardiovascular Society, 2023). 1.5 Hypothesis and Aims Given the abundance of confusing literature regarding OXLAMS, specifically 9 and 13-HpODE and HODEs, coupled with the increasing rates of cardiovascular disease beginning with cardiac fibrosis, this project focused on investigating, growing the body of literature, and connecting these knowledge gaps through two aims: ▪ Investigate the effects of LA on mitochondrial health, and fibrosis. ▪ Explore how OXLAMs impact mitochondrial functioning and fibrosis. The contribution of OXLAMS contribute to mitochondrial redox status and fibrosis was investigated by using fibroblasts in vitro. We hypothesized that OXLAM production would be detrimental to mitochondrial functioning as there will be increased ROS production and therefore impact fibrosis by increasing collagen production. 21 Chapter 2: The fibrotic implications of oxidized linoleic acid metabolites 2.1 Overview Due to the overwhelming increase of omega-6 PUFA consumption and supporting literature linking it to non-communicable diseases such as cardiovascular disease and obesity, we decided to further investigate how LA, the primary parent metabolite of omega-6 PUFAs, can impact the development of fibrosis. This area of research investigating oxidized LA metabolites (OXLAMs) is heavily studied yet conclusions are conflicting surrounding the effects of OXLAMs (Vangaveti et al., 2010) (Nagy et al., 1998) (Hamblin et al., 2021). In vitro studies performed with NIH/3T3 fibroblasts underwent LA dosing and 15-LOX inhibition to prevent 13-HpODE formation (Snodgrass and Brüne, 2019). Mitochondrial redox potential was looked at to determine the effects of the treatment. Following this, collagen assays were performed to determine 13-HpODE’s impact on collagen production. Gene expression analysis through qPCR followed this so that the type of collagen impacted could be determined. Since LA is known to be detrimental to the homeostasis present with collagen production in fibroblasts, it was expected that inhibiting some of its downstream metabolites would benefit the fibroblast and limit the pro-fibrotic phenotype that LA causes (Beam et al., 2015). Lastly, following the gene expression results of glutathione peroxidase-1 and 4, we pursued investigation into superoxide dismutase and catalase activity as well as glutathione supplementation to determine if any negative impacts could be restored. hi 2.2 Materials and Methods 2.2.1 In vitro cell model NIH/3T3 mouse-derived fibroblasts were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). Fibroblasts were cultured in Dulbecco’s Modified Eagle Medium with High Glucose (DMEM; Hyclone, Logan, UT, USA, Cytiva No.SH30022.01) with 10% bovine calf serum (BCS) (Cytiva No. SH30073.03HI), sodium pyruvate (2mM), and 2% penicillin/streptomycin at 37ºC in a humidified atmosphere of 5% CO2. Cells were grown in Corning T75 cm2 flasks and passaged at a maximum of 80% confluency. Cells were treated with 0.25% (w/v) Trypsin-EDTA- 0.53 mM EDTA 22 solution, centrifuged, resuspended, and plated at 10,000 cell/well in clear 48-well plates from Cell Star. Cells were used between passages 4-10. 2.2.2 Reagent and Treatment Preparations Fatty Acid ethyl esters (linoleic acid (LA), and oleic acid (OA)) were purchased from Cayman Chemicals and 5mM stocks were made up with fatty-acid free bovine serum albumin (BSA) (Sigma: A7030-10G). Working stocks were made up and cells were incubated for 24 hours at doses of either 0.1, 0.5, 1.0, or 1.5mM LA or OA. BSA was used as a control for these experiments. For experiments that required multiple treatments, working concentrations and volumes were adjusted accordingly. 9-HpODE (Cayman: 10705) and 13-HpODE (Cayman Chemicals:10704) stocks were made up with reagent grade ethanol (EtOH) and cells were treated with doses of 30uM, using EtOH as the control. 9(S)-HODE (Cayman: 38400) and 13(S)-HODE (Cayman Chemicals: 38600) stock solutions were made up by dilution in reagent grade ethanol (EtOH) and cells were treated with doses of 0.1, 1, 5, 10, and 30uM, using EtOH as the control. After initial experiments, 30uM was used as the working concentration with 9- and 13-HODEs. A potent 15-lipoxygenase inhibitor, 9c(i472) (Cayman Chemicals:28225) was also used in cell treatments. 9c(i472) is a stereospecific and highly selective 15-LOX inhibitor, with a half maximal inhibitory concentration (IC50) of 0.19μM, the most specific and successful in inhibition when compared to its analogs (Guo et al., 2019). The inhibitor was diluted in dimethyl sulfoxide (DMSO) and cells were treated at a concentration of 0.1uM. For some experiments, reduced glutathione (GSH) working stock was made up to 2mM with sterile cell water and co-incubated with LA with or without 9c(i472). When HpODE and HODE activity was investigated, hydrogen peroxide (H2O2) at a dosing concentration of 2mM was also made up with sterile cell water. Cells were co-incubated with 2mM H2O2 ± 2mM GSH and 9- or 13- HpODE or 9-or 13-HODE for 24 hours. Working stocks were manipulated so that the final concentrations were consistent throughout experimentation and between treatments. All reagents were sterilely diluted 23 and stored at the appropriate temperatures between experiments. Fatty acids and HpODEs were stored at -80ºC, HODEs and 9c(i472) at -20ºC, GSH at 4ºC, and H2O2 at room temperature. 2.2.3 MTT Assay The MTT assay is a qualitative and quantitative assay that measures changes in mitochondrial redox potential which may indicate stress in the mitochondria (Mosmann, 1983). It is commonly used as an approximation of cell viability (Mosmann, 1983). It uses the water-soluble tetrazolium dye reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) which is reduced to an insoluble purple product (Mossman, 1983). After 24 hours of incubation with FA treatment, 12mM MTT (made up with PBS) was added to each well of a 48-well plate at a final concentration of 0.5mg/ml and incubated at 37ºC for 1 hour. Formazan crystals were dissolved by adding a volume of sodium dodecyl sulfate (20% w/v)/ N,N-dimethylformamide (50% w/v) solution at a 1:1 volume per well. After aspirating, samples were transferred to a 96-well plate and absorbance read at 570nm on BioTek Cytation 7 Cell Imaging Multimode Reader (Agilent, Santa Clara, CA, USA) and analyzed by the Gen5 software (Agilent, Santa Clara, CA, USA). 2.2.4 Picosirius Red Staining Sirius Red is a strong stain for extracellular collagen products such as type I and III collagen, relevant to this project (Beam et al., 2015). A protocol was adapted from Tullberg-Reinert and Lundt (H Tullberg-Reinert & Jundt, 1999), Beam et al., (Beam et al., 2015), and the SirCol reagent to obtain soluble and deposited collagen results. Soluble and Deposited Collagen assays were done for all conditions of treatments described in 2.2.2 above. 2.2.4.1 Soluble Collagen Assay After 24 hours of treatments described in Section 2.2.2, 50ul of cell medium from each treated well was added to 50ul of sterile cell water in 1.5ml tube to reduce serum concentration to 5% (Szász et al., 2023). Reducing the serum concentration reduces non-collagenous proteins present in serum that may interfere with Sirius red (Szász et al., 2023). 250ul of Sirius Red dye (Abcam: ab247832) was added to each tube and mixed gently at room temperature for 30 mins. Tubes were centrifuged for 5min at 10000g 24 at 4ºC and supernatant removed. The pellet was resuspended in 0.01N HCl to remove the unbound dye and spun at the same parameters above once more. HCl was removed and 250ul of 0.1N NaOH added. Samples were transferred to a new 48-well plate and Absorbance immediately read at 540nm on the BioTek Cytation 7 Cell Imaging Multimode Reader (Agilent, Santa Clara, CA, USA) and analyzed by the Gen5 software (Agilent, Santa Clara, CA, USA). 0.1N NaOH blank was subtracted, and samples were normalized to the control treatment depending on the experiment. 2.2.4.2 Deposited Collagen Assay After fibroblasts were treated (as described in 2.2.2), treated wells were washed twice with PBS and cells were fixed with 100ul of Bouin’s fixative (Sigma: #HT10132) for 1 hour at room temperature. The fixative was removed under running water and plates were air dried. 200ul of Sirius Red dye was then added to each well and the plate incubated for 1 hour at room temperature under mild shaking. Excess dye was carefully removed, and cells washed three times with 0.01N HCl. Photos were taken at this point in the assay. The stain was then dissolved in 200ul of 0.1N NaOH under gently shaking at room temperature for 1 hour. Absorbance was measured at 540nm with 0.1N NaOH as a blank on the BioTek Cytation 7 Cell Imaging Multimode Reader (Agilent, Santa Clara, CA, USA) and analyzed by the Gen5 software (Agilent, Santa Clara, CA, USA). Samples were normalized to the control treatment for the respective experiment. 2.2.5 RNA extraction, cDNA synthesis, and qPCR To extract RNA from cell culture fibroblast samples treated as described in Section 2.2.2, the following protocol was adapted from Invitrogen™ TRIzol™ (ThermoFisher: No. 15596026) reagent protocol as follows. Treated wells were washed with ice cold PBS and 100ul of TRIzol™ was added per well and left for 5min at room temperature. After transferring samples to 1.5ml tubes, 20ul of chloroform was then added and the tubes vortexed vigorously then left to sit for 2 minutes at room temperature. Samples were then centrifuged at 12000g for 15 minutes at 4ºC. The aqueous layer was then removed and transferred to a new tube where isopropyl alcohol was added and samples vortexed again. Tubes were then centrifuged again at the same parameters outlined above. A wash was performed with 75% EtOH in 25 RNase free H2O and vortexed. Samples were again centrifuged at 7500g for 5min at 4ºC. The supernatant was removed and redissolved in RNAse free H2O. Samples were normalized to 50ng/ul and used for cDNA synthesis. cDNA synthesis was performed using OneScript Hot cDNA synthesis kit (ABM Inc., Richmond, BC, Canada) To measure gene expression, qualitative real-time polymerase chain reaction (qPCR) was utilized and performed on the Bio-Rad CFX 2.0 Machine (BioRad). Ssofast Evagreen Supermix (BioRad: #1725200) was used for gene amplification and all desired primers synthesized by Integrated DNA Technologies (IDT Coralville, Iowa, USA). Samples were run in duplicates with a final reaction volume of 10ul. PCR was performed under the following conditions: 95ºC for 30sec, 50 cycles of 95ºC for 5sec, 58ºC for 5sec, 95ºC for 10sec, followed by a melt curve analysis at 65ºC-95ºC increasing by 0.5ºC every 5sec. CFX Manager software version 3.1.1517.0823 was used to quantify gene expression with 18s and β-actin used as reference genes for all reactions. Relative normalized gene expression was quantified with the ΔΔCt method using treatment-respective control groups. The following equations were then used on the log transformed data: 𝛥𝐶𝑞 = 𝐶𝑞 (𝑡𝑎𝑟𝑔𝑒𝑡 𝑔𝑒𝑛𝑒) − 𝐶𝑞(𝑟𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒 𝑔𝑒𝑛𝑒) 𝛥𝛥𝐶𝑞 𝑒𝑥𝑝𝑟𝑒𝑠𝑠𝑖𝑜𝑛 = 𝛥𝐶𝑞(𝑠𝑎𝑚𝑝𝑙𝑒) − 𝛥𝐶𝑞(𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑔𝑟𝑜𝑢𝑝 𝑎𝑣𝑒𝑟𝑎𝑔𝑒) 𝐹𝑜𝑙𝑑 𝐶ℎ𝑎𝑛𝑔𝑒 = 2−(𝛥𝛥𝐶𝑞) 26 Table 2.1: qPCR Primer Sequences Gene Sequence (5’→ 3’) 18s R: GCTGGAATTACCGCGGCT F: CGGCTACCACATCCAAGGAA Β-actin R: CATGCCCACCATCACGCCCTGG F: ACAGAGCCTCGCCTTTGCCGA Collagen 1 R: GACTGTTGCCTTCGCCTCTG F: ACCTGTGTGTTCCCTACTCA Collagen 3 R: TGGGGTTTCAGAGAGTTTGGC F: AATGGTGGTTTTCAGTTCAGC GPx1 R: CTTCGGACACCAGGAGAA F: GCCTTCTCACCATTCACTTC GPx4 R: GTGGGCATCGTCCCCATTTA F: GGCAGGAGCCAGGAAGTAAT 2.2.6 Superoxide Detection assay MitoSoX™ Red (Invitrogen™: M36008) is a fluorescent indicator that is oxidized by superoxide systems and no other ROS systems in the mitochondria. Once oxidized, it will produce a red fluorescence which can be quantified under 510/580nm excitation/emission maxima. The following protocol was adapted for a 48-well plate from the MitoSoX™ Red product manual. In the dark, each well of fibroblasts treated with 30uM HpODE or HODE with or without 2mM GSH was washed once with PBS. Then, after adding 190ul of NIH/3t3 media, 10ul of 5mM MitoSoX™ stock was added to each well and then incubated for 10min at 37ºC. Wells were washed once with warm PBS, counterstained with Hoechst 33342 (ThermoFisher), and fluorescence was read on the BioTek Cytation 7 Cell Imaging Multimode Reader (Agilent, Santa Clara, CA, USA) and analyzed by the Gen5 software (Agilent, Santa Clara, CA, USA). 27 2.2.7 Statistical Analysis GraphPad Prism 9 was used for all statistical analyses. One-way or two-way ANOVAs were used to determine if data sets were significant when compared to control or when compared to one another, respectively. Šídák’s or Dunnet’s multiple comparisons post-hoc tests were conducted when appropriate. All data was represented as mean ± standard error of the mean (SEM) and used a probability (p) value of less than 0.05 to indicate significance. Significance is both reported in figures, denoted by *, **, ***, or **** representing p<0.05, p<0.01, p<0.001, and p<0.0001, respectively, above bar graphs as well as stated in the figure captions. Sample size (n) is also represented in the figure captions. 2.3 Results 2.3.1 Linoleic Acid reduces mitochondrial redox potential when compared to oleic acid. To determine the effects of LA on mitochondrial redox potential, MTT assay was completed. After incubation for 24 hours with varying doses of either linoleic or oleic acid, fibroblasts underwent an MTT assay which produced results indicative of mitochondrial redox potential (Figure 8). At lower doses (0.1mM and 0.5mM) fibroblasts did not have any changes in mitochondrial redox potential with LA but at higher doses (1.0 and 1.5mM) mitochondrial redox potential faced a significant decrease of p<0.001 28 and p<0.0001, respectively, when compared to BSA alone (vehicle). In contrast, oleic acid had increases in redox potential at 0.5 and 1.5mM, (p<0.05). Figure 8: Changes in mitochondrial redox potential of NIH/3T3 fibroblasts after 24 hours of incubation with varying doses (0.1, 0.5, 1.0, 1.5mM) of either linoleic or oleic acid. Significance indicated on figure by * (p<0.05) in a Two-way ANOVA with a Dunnet’s post-hoc test. Experimental groups had a sample size of n=3. 2.3.2 Linoleic acid increases soluble and deposited collagen production. To determine if the drop in mitochondrial redox potential with higher LA doses could be causing changes in fibrotic response, fibroblasts underwent soluble and deposited collagen Sirius red staining. Following 24 hours of incubation with varying doses of oleic or LA (as described in 2.2.2), fibroblasts were then imaged (Figure 11) and subsequent readings at OD540nm was recorded to show that there was a significant (p<0.05) increase in soluble collagen production in 1.5mM LA treatment groups (Figures 9 & 10). Images were used for qualitative purposes only. Deposited collagen is identified in Figure 11, as Picrosirius red stain is not cell permeable, deposited collagen fibers are bound by the red dye in the ECM outside of the cell (eg. on top of adhered cell) (Junqueira et al., 1979) (Tullberg-Reinert & Jundt, 1999) (Chen et al., 2012) (Chaubaroux et al., 2015). Although the lower doses were not significant, the 29 beginnings of an upwards dose-dependent trend can be seen in soluble collagen production with LA. Oleic acid doses did not influence soluble collagen production. Deposited collagen production was recorded to be insignificant although at lower doses of LA treatment (0.1mM) there was a slight statistically insignificant increase in raw values. (Figure 10). Figure 9: Soluble Collagen Assay of fibroblasts after 24 hours of incubation with varying doses (0.1, 0.5, 1.0mM) LA or OA. Significance indicated on graph with * (p<0.05) and obtained through Two-way ANOVA with a Šídák’s post-hoc test. Experimental groups had a sample size of n=3. Figure 10: Deposited Collagen Assay of fibroblasts after 24 hours of incubation with varying doses (0.1, 0.5, 1.0mM) LA or OA. Results were statistically insignificant and were obtained through Two-way ANOVA with a Šídák’s post-hoc test. Experimental groups had a sample size of n=3. 30 Figure 11: Representative images of Sirius Red staining of deposited collagen in BSA (vehicle control) treated fibroblasts (A) when compared to 1.0mM LA treated fibroblasts (B). Magnification 40x. Images taken on EVOS Cell Imaging Microscope (ThermoFisher). White arrows indicate examples of deposited collagen in ECM. 2.3.3 Inhibiting 13-HpODE production at high LA doses restores mitochondrial redox potential and increases deposited collagen formation. To investigate the role of OXLAMS derived from LA, fibroblasts were incubated with varying doses of LA (0.1, 0.5, 1.0, 1.5mM) and co-treated with 0.1uM 9c(i472), a 15-LOX inhibitor which specifically inhibits 13-HpODE production (Figure 12). At low doses, there are no significant changes among groups, but at higher doses (>1.0mM), 9c(i472) caused a significant increase and restoration of mitochondrial redox potential, when compared to control (DMSO or BSA). Downstream stable OXLAMs (9- and 13-HODEs) had no impact on mitochondrial redox potential (Appendix A) and were not investigated further after this result. In a similar manner as described in Section 2.3.2, collagen production with inhibited 15-LOX was explored. Results for deposited collagen nearly mirrored those of the MTT assay where deposited collagen production increased at higher doses (0.5 and 1.0mM) when cells were co-treated with LA and 0.1uM 9c(i472) (Figure 13). Soluble collagen under these conditions showed no significance between A B 31 groups raw data presented increases in soluble collagen when 13-HpODE production was inhibited at lower doses of LA (Figure 14). Figure 12: Changes in mitochondrial redox potential of NIH/3T3 fibroblasts after 24 hours of incubation with varying doses (0.1, 0.5, 1.0, 1.5mM) of linoleic acid ± 0.1uM 9c(i472). Significance indicated on figure by **** (p<0.0001) and obtained through a Two-way ANOVA with a Šídák’s post-hoc test. Experimental groups had a sample size of n=3. Figure 13: Deposited Collagen Assay of fibroblasts after 24 hours of incubation with varying doses (0.1, 0.5, 1.0mM) LA with or without 0.1uM 9c(i472). Significance indicated on graph with * (p<0.05) and obtained through Two-way ANOVA with a Šídák’s post-hoc test. Experimental groups had a sample size of n=3. 32 Figure 14: Soluble Collagen Assay of fibroblasts after 24 hours of incubation with varying doses (0.1, 0.5, 1.0mM) LA with or without 0.1uM 9c(i472). Results were statistically insignificant and were obtained through Two-way ANOVA with a Šídák’s post-hoc test. Experimental groups had a sample size of n=3. 2.3.4 Inhibiting 13-HpODE increases Collagen III and decreases Collagen I gene expression dose-dependently. Gene expression of Collagen Types I and III was determined through qPCR after observing the increase in deposited collagen with the Sirius red assay (Figure 13) when 13-HpODE was not synthesized. When compared to LA only treated fibroblasts, qPCR results showed significant increases in collagen III expression at all doses of LA treated and inhibited fibroblasts. Collagen I expression showed significant increases at lower (0.1 and 0.5mM) doses of inhibited LA treatments when compared to LA-treatment but had limited expression with higher doses of LA, with or without inhibition. 33 Figure 15: Gene expression. Left: Collagen 3 gene expression of LA treated fibroblasts with or without 15-LOX inhibition. Right: Collagen I gene expression of La treated fibroblasts with or without 15-LOX inhibition. Results were statistically significant and obtained through two-way ANOVA with a Šídák’s post-hoc test. Experimental groups had a sample size of n=3. 2.3.5 Oxidatively stressed fibroblasts treated with HpODEs significantly reduces redox potential which cannot be restored with GSH supplementation. Since 15-LOX inhibition provided significant results regarding the role 13-HpODE may play in collagen production and gene expression, it was pursued further. While 30uM of 9- or 13-HpODEs did not cause a significant change in mitochondrial redox potential, inducing oxidative stress with the addition of 2mM H2O2 did. There was a significant decrease in mitochondrial redox potential 30uM 9-HpODE with 2mM H2O2 and an even larger decrease when the same conditions were applied with 30uM of 13-HpODE (Figure 16). With HpODEs being reactive intermediates, and the substrate for GPx4, 2mM GSH was co-incubated with 30uM of 9- or 13-HpODE to determine if this could possibly increase flux through the GPx4 conversion pathway to stable 9- and 13-HODE products, respectively. As seen in Figure 16, these conditions did not have significant effects on mitochondrial redox potential. Following this, HpODEs were then co-incubated with 2mM H2O2 and 2mM GSH. In the 4th set of bars in Figure 16, a slight yet significant decrease in mitochondrial redox potential for 9-HpODE when compared to 30uM of 9-HpODE alone was seen. An even larger decrease in redox potential was seen with the fibroblasts 34 treated with 13-HpODE, 2mM H2O2, and 2mM GSH indicating 2mM GSH was not able to restore mitochondrial antioxidant reserves in either scenario. Figure 16: Changes in mitochondrial redox potential of NIH/3T3 fibroblasts after 24 hours of incubation with 30uM 9-or 13-HpODE ±2mM H2O2, ±2mM GSH, or both, H2O2 and GSH. Significance indicated on figure and obtained through a Two-way ANOVA with a Šídák’s post-hoc test. Experimental groups had a sample size of n=3. Collagen production was then investigated under the same conditions as seen above, and although insignificant results were produced, 13-HpODE, independently of co-treatment, had higher RFU values than 9-HpODE produced, for both soluble and insoluble collagen (Figures 17 and 18). 35 Figure 17: Soluble Collagen Assay of fibroblasts after 24 hours of incubation with 30uM 9-or 13-HpODE ±2mM H2O2, ±2mM GSH, or both, H2O2 and GSH. Results were statistically insignificant and were obtained through Two-way ANOVA with a Šídák’s post-hoc test. Experimental groups had a sample size of n=3. Figure 18: Deposited Collagen Assay of fibroblasts after 24 hours of incubation with 30uM 9-or 13-HpODE ±2mM H2O2, ±2mM GSH, or both, H2O2 and GSH. Results were statistically insignificant and were obtained through Two-way ANOVA with a Šídák’s post-hoc test. Experimental groups had a sample size of n=3. 2.3.5.1 9- and 13-HODE bioconversion pathways may be governed by different antioxidant systems. Due to the discrepancy in size of decreases in redox potential between 9-HpODE and 13-HpODE co-treated with H2O2, and the fact that GSH was unable to restore this, I hypothesized that these two pathways are governed by different antioxidant systems. GPx1 was first explored with increased expression shown at low doses of LA (0.1mM) and decreased expression at higher doses of LA (0.5 and 1.0mM), relative to control (Figure 19a). When 9c(i472) was added and 15-LOX was inhibited (i.e., 13-36 HpODE not made), all doses of LA showed no activity of GPx1. Next, GPx4 was looked at. At low (0.1mM) doses of LA, there was decreased gene expression when compared to control (BSA). At higher doses of LA, there was no expression of GPx4 when compared to control (Figure 19b). We hypothesized this could be to “stunting” at the GPx4 step due to accumulation of 13-HpODE (the substrate) or diminished GSH stores, necessary for GPx4 to function. 2mM GSH was supplemented to overcome this potential block and the same results as without the GSH supplementation were seen, decreased expression of Gpx4 at lower doses of LA (0.1mM) with 2mM GSH and no expression at doses 0.5mM and 1.0mM LA (Figure 19c). GPx4 gene expression results when 9c(i472) was added to varying doses of LA showed increased GPx4 expression at higher doses (0.5 and 1.0mM) of LA and no expression at lower doses of LA (0.1mM), when compared to control. Since GPx1 and GPx4 gene expression results were not sufficient in connecting a pathway with the outlined conditions, qPCR was used again to determine if catalase may be involved. Insignificant results were found with no expression present with any doses of LA (with/without inhibitor). MitoSoX™ and Hoescht 33342 was utilized to determine if superoxide dismutase (SOD) activity could be detected when fibroblasts were treated with 30uM 9- or 13-HpODE, or 9- or 13-HODE. All results were insignificant and presented as RFU per nuclei in Figure 20. MitoSoX™ 37 was also used to see if inhibiting 15-LOX activity with 9c(i472) would activate the SOD pathway, and again, insignificant results were found. Figure 19a-c: (A) GPx1 expression of LA treated fibroblasts. (B) GPx4 expression of LA treated fibroblasts with or without 0.1uM 9c(i472). (C) GPx4 expression of LA and GSH treated fibroblasts. Results expressed as log transformed relative expression to control. **** indicates p<0.0001 obtained with Two-way ANOVA with Šídák’s multiple comparison test, Groups had a sample size of n=3. 38 Figure 20: Relative fluorescence per nuclei normalized to control (EtOH vehicle) displaying SOD activity. 9- and 13- HpODEs and 9-, and 13-HODEs were all dosed at 30uM. All experiments n=3 and obtained using MitoSoX™ dye and counterstaining with Hoescht 33342. Results were insignificant and obtained through Two-way ANOVA with a Šídák’s post-hoc test. Groups had a sample size of n=3. 2.3.6 GSH supplementation restores mitochondrial redox potential with high doses of LA. Although 2mM GSH supplementation was not able to restore redox potential in H2O2 and 9- or 13-HpODE treated fibroblasts, it was able to restore mitochondrial redox potential in LA-treated fibroblasts significantly at high (1.5mM) doses (Figure 21). Figure 21: Changes in mitochondrial redox potential of NIH/3T3 fibroblasts after 24 hours of incubation with varying doses of LA (0.1, 0.5, 1.0, 1.5mM) with or without 2mM GSH. Significance indicated on figure and obtained through a Two-way ANOVA with a Šídák’s post-hoc test. Groups had a sample size of n=3. 2.4 Discussion The North American diet has been under continuous scrutiny for the past few decades (Patterson et al., 2012) for promoting non-communicable diseases. While industrial-level changes would need to be implemented for populations to truly feel an impact, this research set out to contribute to the body of 39 literature regarding a link between increased omega-6 consumption (specifically, LA) and fibrosis in cardiovascular disease. The novel aspect of this thesis involves the use of OXLAMS directly rather than the parent LA as the stressor of cells, thereby removing other potential confounding effects of multiple metabolites of LA. NIH/3T3 fibroblasts are used for investigating mechanisms involving fibrosis as well as fatty acid metabolism (De et al., 1994). After treatments with fatty acids, inhibitors, and supplementation with GSH or H2O2, an MTT assay was utilized to determine the impact of outlined treatments in 2.2.2 on mitochondrial redox potential. MTT assay has been used previously for cell viability studies involving NIH/3T3 cells (Danihelová et al., 2013). After 24 hours of incubation with LA or OA, a significant decrease in redox potential was seen at high (1.0 and 1.5mM) doses of LA. This was as expected based on literature showing detrimental effects in terms of mitochondrial function when LA is in excess (Schuster et al., 2018), (Jin et al., 2021). Excess dietary LA and increased ROS production contributing to mitochondrial dysfunction is a common link between these studies and plays a role in subsequent development of chronic inflammation (Heller et al., 2003) (Pahwa et al., 2023) (Ramsden et al., 2010). As in our lab’s last in vivo study reporting that LA increased fibrosis, we now re-investigated how LA impacts collagen production (Beam et al., 2015). There was an increase in soluble collagen formation as the dose of LA increased. Excess production of soluble collagen is a stress response indicating mitochondrial stress and/or dysfunction due to the cell changing its ECM makeup (Ricard-Blum, 2010), (Plikus et al., 2021). This was seen with a decrease in redox potential through the MTT assay for LA treatment (Figure 8). Since there were changes seen in the physical products of soluble or deposited collagen, gene expression of the two most common types of collagens (I and III) were investigated. Relative to one another, collagen I expression was increased, and collagen III decreased with LA incubation. LA is a known stimulator of a pro-fibrotic phenotype amongst fibroblasts, through activation of TGFβ1 and so, such alterations in the collagen I to III ratio was expected (Rosenkranz, 2004) (Beam et al., 2015). The results obtained here supported the hypothesis that at higher doses of LA (0.5mM and above), fibroblasts demonstrated mitochondrial dysfunction and promote a pro-fibrotic phenotype. 40 Interestingly, no collagen gene expression was seen at higher doses of LA (0.5mM and above) probably indicating cytotoxic effects of LA preventing fibroblasts from continued gene expression of either collagen type. Such an effect was recorded earlier in our lab (Beam et al., 2015). To explore the effects of downstream LA bioconversion to OXLAMs, the most abundant final products, 9- and 13-HODE, were investigated (Vangaveti et al., 2010). Independent treatments of 0.1 to 30uM of 9-HODE or 13-HODE in fibroblasts saw no changes in mitochondrial redox potential over a 24-hours treatment (Appendix A). This was surprising considering that extracellular concentrations of 13(S)-HODE were shown previously to induce mitochondrial dysfunction and detrimental structural changes in airway epithelial cells (Mabalirajan et al., 2013). Studies have shown that 13(S)-HODE is often taken up into membranes as phosphatidylcholine, although this incorporation only occurred for the first 6-8 hours of incubation. As this project examined the impact of 13-HODE over 24 hours (Fang et al., 1999), the absence of an impact might indicate an adaptation to increased 13-HODE over longer periods in these dividing cells. In a few in vivo studies conducted with OXLAMs, mice fed OXLAM-rich diets experienecd hepatocyte cell death (Schuster et al., 2018). This study utilized 9-HODE and 13-HODE as well as oxo-ODEs as well as hydroxy-eicosapentaenoic acids (HEPE) (Schuster et al., 2018). LA is first converted to 13-HpODE by 15-LOX, and then to its stable metabolite, 13-HODE, by GPx4 in the presence of GSH. Whereas LA is converted to 9-HpODE through 5-LOX and non-enzymatic mechanisms and then to 9-HODE in the same manner as 13-HODE. To investigate how LA’s bioconversion to 13-HODE impacts mitochondrial function, 15-lipoxygenase1 was inhibited with 0.1uM 9c9(i472). Intriguingly, addition of this inhibitor restored the LA mediated drop in mitochondrial membrane potential, indicating that the LA mediated loss in mitochondrial membrane potential is primarily due to 13-HpODE synthesis (Figure 12). This is because 13-HpODE as a reactive intermediate depletes mitochondrial antioxidant reserves to cause oxidative stress. These findings suggest that 13-HpODE may be detrimental to mitochondrial health and that inhibition of its synthesis could benefit mitochondrial functioning. 41 In terms of collagen production, inhibition of 13-HpODE did not alter the levels (p=0.0858) of soluble collagen at any dose of LA. However, deposited collagen displayed correlated with MTT results, i.e. there was an increase in deposited collagen formation at 0.5 and 1.0mM doses of LA treated fibroblasts when 15-LOX was inhibited. As collagen deposition follows formation of soluble collagen, it means that oxidative stress was tolerable within the mitochondria, and all extra soluble collagen produced under 13-HpODE inhibition was able to be converted to deposited collagen. As mentioned earlier, LA (amongst other omega-6 PUFAs) is known to be a promoter of a pro-fibrotic phenotype within fibroblasts (Dai et al., 2011), (Gibb et al., 2020), (Saadat et al., 2021). However, this experiment did demonstrate that the rate of collagen production by be attenuated or the conversion of soluble to deposited collagen may be accentuated with an inhibition of 13-HpODE synthesis. Following the results seen in collagen production and the restoration of mitochondrial redox potential when 13-HpODE was inhibited, gene expression of various collagen isoforms was evaluated. At lower doses of LA (0.1mM) inhibited with 9c(i472), a sharp increase in collagen I expression was seen compared to LA alone, showing us that 13-HpODE must be an important regulator in collagen I production at low levels of LA. Conversely, expression of collagen I was decreased at a higher dose of LA (0.5mM) with the inhibitor. This was accompanied by increased expression of collagen III (Figure 15) at any dose of LA with inhibitor when compared to LA-treated fibroblasts alone. It appears that inhibiting the bioconversion of LA to 13-HpODE increases collagen III signaling in fibroblasts. Our lab has shown that LA itself can alter the collagen I:III ratio in favour of “stiffening” the heart tissue by increasing collagen I (Beam et al., 2015). These results indicate that 13-HpODE may act as a regulator for this ratio, inhibiting collagen III production, which forms the basis of this altered collagen ratio. Following these discoveries, we hypothesized that these results (specifically the detrimental ones seen with LA could be due to a possible buildup in the intermediate step (HpODEs) and the lack of flow through GPx4, limited by the GSH concentration. Since HpODEs are reactive intermediates and get reduced to stable HODEs, they require a properly functioning GPx4 with excess GSH (Figure 5) (Stolwijk et al., 2020). This began our experimentation with GSH supplementation to restore these 42 depleted antioxidant reserves, allowing for greater flux of HpODE to HODE bioconversion. After confirming that the working dose of both 9- and 13-HpODE did not impact mitochondrial function (i.e., this dose of HpODE did not exceed the mitochondria and GPx4’s capabilities to neutralize it), 2mM of GSH was added to see if there would be any changes to redox potential. No significant differences were observed with GSH supplementation in mitochondrial membrane potential. Oxidative stress is known to cause mitochondrial dysfunction. To assess how mitochondria could handle the flux of HpODE coming from LA, 2mM H2O2 was used next to induce oxidative stress (Ransy et al., 2020) (Figure 16). We hypothesized that both 9- and 13-HpODE will undergo the same decreases in redox potential when the mitochondria are oxidatively stressed as GSH stores will now have to be shared between GPx1 (neutralizes H2O2) and GPx4 (neutralizes HpODE). While both did experience a decrease in mitochondrial potential, the co-incubation of 13-HpODE and H2O2 caused a significantly damaging drop in mitochondrial redox potential when compared to 9-HpODE and H2O2. We concluded that 13-HpODE depletes antioxidant reserves at a greater extent than the 9-HpODE pathway. Next, we evaluated the gene expressions of GPx1 and Gpx4. A low dose of LA (0.1mM) increased GPx1 expression (Figure 19a), but no expression was detected at higher doses (0.5 and 1.0mM). Such lack of change in GPx1 for LA-treated cells is supported by the fact that GPx1 is involved in reducing H2O2, not hydroperoxides (Brigelius-Flohé & Maiorino, 2013). However, the increase in GPx1 expression with a low dose of LA (i.e. 0.1mM) was indicative of an induction of antioxidant enzymes with low levels of oxidative stress and has been termed as antioxidant hormesis (Nitti et al., 2022) (Valaei et al., 2021). LA is a known inducer of oxidative stress. Inhibiting the synthesis of 13-HpODE increased GPx4 gene expression. Expectedly, at low doses of LA and 15-LOX inhibitor, there was no expression of GPx4 indicating that this enzyme was not needed yet. At 0.5mM and 1.0mM of LA, with the inhibition of 15-LOX, GPx4 expression was increased. This could suggest that the mitochondria at these higher doses (0.5 and 1.0mM) of LA, in the presence of the 15-LOX inhibitor, was preparing itself to combat other metabolites of LA including ARA, HETEs or 43 9-HpODE. Without doing a detailed analysis of lipid metabolites at this exact stage, such ideas remain a speculation. The mitochondria utilize three main antioxidant systems to eradicate oxidative stressors: glutathione peroxidases (with GPx1 responsible for H2O2 neutralization and GPx4 for hydroperoxides), superoxide dismutase (SOD, for neutralization of superoxide radicals) and finally, catalase (for metabolizing H2O2) (Litwack, 2018). Gene expression showed insignificant results for catalase and although at first surprising, data in the literature supported this. Studies reported that there may be transient activation of catalase with 13-HpODE and 13-HODE treatment, but no activity at the 24-hour treatment mark (Faizo et al., 2021). MitoSoX™ was used for superoxide detection (Figure 20) and insignificant results were seen for both 9- and 13-HpODE and HODEs. Although statistically insignificant, 13-HpODE and 13-HODE did have higher RFU values in general than 9-HpODE and 9-HODE. Both soluble and deposited collagen results showed insignificant trends when co-incubated treatments (either 2mM GSH, 2mM H2O2, or both) were compared to 30uM of HpODE alone (Figures 17 and 18). While statistically insignificant, one trend was seen. 13-HpODE for both soluble and deposited collagen production produced higher RFU values than 9-HpODE. This was consistent with the results seen in the MTT assay, where 13-HpODE when co-incubated with 2mM H2O2 or 2mM GSH presented significant decreases in mitochondrial redox potential indicating mitochondrial dysfunction. This allowed us to conclude that when 13-HpODE is present with another stressor (in this case, H2O2), the mitochondria are unable to maintain homeostasis and combat the oxidative stress. This in turn, leads to dysregulation of ECM maintenance, seen with larger amounts of collagen production. 44 Chapter 3: Conclusion 3.1 Significance of Findings There is a lot of literature that supports the impact that excess LA consumption on non-communicable diseases and chronic inflammation (Beam et al., 2015) (Heller et al., 2003) (Lee et al., 2022). Although these studies have shown how LA itself may impact downstream physiological effects, such as the development of fibrosis, limited studies show how HpODEs and HODEs play a role in the mechanisms of fibrosis and its contribution to cardiovascular disease (van Leyen et al., 1998), (Kühn & Brash, 2020). The findings of this thesis are significant for several reasons. The first being that to our knowledge, there are no studies that have investigated how the formation or lack thereof of reactive intermediates (HpODEs) from excess LA impacts the type of collagen produced. Since LA is known to increase Type I collagen formation (Beam et al., 2015), these results showed that the bioconversion to 13-HpODE is essential to inhibiting collagen III formation and necessary for collagen homeostasis in the context of fibrosis. The second reason of significance is that it has been shown that chronic inflammation is caused by mitochondrial dysfunction, typically stemming from chronic oxidative stress (Pahwa et al., 2023). The results shown in Chapter 2 demonstrates that 13-HpODE depletes antioxidant reserves in an oxidatively stressed mitochondria at a greater rate than 9-HpODE. We believe that active inhibition of the formation of 13-HpODE could be seen as a therapeutic target to address ‘stiff’ myocardium in heart disease, especially in context to the Western diet, where individuals are exposed to high amounts of LA in their diet. Finally, there were promising results showing how 2mM supplementation of GSH (or restoration of healthy physiological levels) can restore the reduction in mitochondrial redox potential seen when fibroblasts were subjected to high doses of LA. This finding along with our earlier findings from our lab continue to suggest chronic antioxidant supplementation as a long-term strategy in preventing oxidative stress in heart disease. 45 3.2 Limitations of research and Future work It is important to note the context of these results. These studies were performed on immortalized mouse fibroblasts, rather than primary human fibroblasts. Although there are many similarities between the two, mechanistically, there are still many physiological limitations to using mouse-derived cells, rather than human-derived cells (Rydell-Törmänen and Johnson, 2019). Mouse-derived fibroblasts were also used to lay the groundwork for future in vivo studies. Another limitation was that 5-lipoxygenase (5-LOX) was not investigated. This enzyme is responsible for the bioconversion of LA to 9-HpODE. This was not pursued for two reasons; one being time and resources, and the second being that 9-HpODE can also be synthesized non-enzymatically alongside 5-LOX. These considerations prevented us from pursuing a detailed analysis of this particular OXLAM, rendering it not feasible. Studies building off these results should include a in vivo study with high and low omega-6 (LA) diets, OXLAM-rich diets, as well as inhibitors to further investigate any therapeutic advantages to inhibiting LA’s bioconversion to HpODEs and subsequent HODEs in in vivo models. This would also provide cardiac tissue to investigate fibrosis development. Supplementation with glutathione may also be something to further explore as the restoration of LA’s detrimental impact on mitochondrial redox potential presented interesting results in vitro. 3.3 Main Conclusions In summary, our hypothesis was able to be accepted as OXLAM production, specifically 13-HpODE, increased ROS production, decreased mitochondrial redox potential and subsequently, impacted collagen production by promoting a pro-fibrotic phenotype by increasing collagen production. 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Thesis/Dissertation
eng
Biochemistry and Molecular Biology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Ghosh, Sanjoy
Investigating the fibrotic effects of oxidized linoleic acid metabolites
Text