Oxidised fatty acid derivatives and their putative receptors in atherosclerosis and diabetes
Vangaveti, Venkat N. (2011) Oxidised fatty acid derivatives and their putative receptors in atherosclerosis and diabetes. PhD thesis, James Cook University.
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Abstract
Atherosclerosis, a disease of the arteries is one of the leading causes of mortality throughout the world. A variety of risk factors contribute to this cardiovascular disease including genetic factors, diet, lack of exercise and other lifestyle changes that have become prevalent during the last few decades. These risk factors contribute to two important characteristic features; increased levels of lipids in the arterial wall and oxidative stress – both leading to accumulation of oxidised lipids. Once modified, lipids are reactive with arterial endothelial cells, vascular smooth muscle cells and macrophages. The latter are one of the primary cell types that are attracted to sites of inflammation in the arterial wall, and are derived from circulating monocytes. Macrophages have immune functions and phagocytic properties enabling them to take up oxidised lipids. These latter molecules are known to be key regulators of atherosclerosis. Understanding the fundamental mechanisms of interaction between oxidised lipids and macrophages might enable us to modulate metabolic processes involved in atherosclerosis. Two components of oxidised lipids are the hydroxyl-derivatives of linoleic acid, derived by action of the enzyme lipoxygenase during early stages, and non-enzymatically during the later stages of atherosclerosis. The products of this oxidation are 9-hydroxyoctadecadienoic acid (9-HODE) and 13- hydroxyoctadecadienioc acid (13-HODE). The nature of their interaction with macrophages and how they influence key processes involved in disease progression are not completely understood. We therefore, tested the hypothesis, in this study, that 9- HODE and 13-HODE would have differing effects on monocytes and macrophages in regulating atherosclerosis. We aimed to test the effects of HODEs and their parent fatty acid linoleic acid and the n-3 fatty acid α-linolenic acid on cell proliferation, differentiation, genes of lipid metabolism and generation of inflammatory mediators in THP-1 monocyte-macrophage cells. We wished to establish whether HODEs might act through the recently described G protein-coupled receptors for long-chain fatty acids GPR120 and GPR132. This study investigates regulation of expression of these receptors in THP1 cells and in primary monocyte cultures from diabetic patients where they were studied alongside serum markers for cardiovascular risk.
HODEs decreased cell viability in both monocytes and macrophages in a dose-dependent manner. This was found to be a result of apoptosis, an effect not seen with α-linolenic and linoleic acids. Gene expression studies revealed increased expression of the intracellular lipid transporter FABP4 in both monocytes and macrophages with HODEs. Also, an increase in expression of scavenger receptor B (decreasing lipid burden) with HODEs in macrophages was observed. These processes were accompanied by an increase in expression of the transcription factor peroxisome proliferated-activated receptor-γ (PPAR-γ), which regulates inflammation. GPR132 was highly up-regulated by HODEs in monocytes, while HODEs decreased expression of GPR120 in macrophages. None of these effects were observed with α-linolenic acid or linoleic acid. Similar effects of HODEs on lipid regulatory genes were documented to occur during macrophage differentiation.
Overall, studies with Oil Red O and gene expression suggested that HODEs enhance processes that are associated with reduction of lipid burden. Further studies with HODEs demonstrated that they are involved in regulating secretion of pro-inflammatory cytokines with effects mediated through both PPAR-γ and GPR132. By decreasing both lipid accumulation and vascular inflammation, HODEs may thus have an overall protective effect as far as atherosclerosis is concerned. The mechanisms underlying these protective effects was investigated using specific PPAR-γ antagonists and by silencing the GPR132 gene. Here, successfully for the first time GPR132 was silenced in THP-1 macrophages. Experiments with HODEs revealed a partial role for PPAR-γ in regulating expression of FABP4, and also that regulation of inflammatory mediators by HODEs was largely GPR132 independent. Expression of GPR132 was also investigated in primary monocytes from diabetic subjects. Mononuclear cells from diabetic subjects expressed higher levels of differentiation markers CD14, CD54 and CD36 (identified using flow cytometry) compared with normal controls. Gene expression studies revealed increased expression of GR132 in monocytes from diabetic subjects. Furthermore, this correlated with increased serum levels of inflammatory proteins, indicating that increased GPR132 might be associated with increased vascular risk.
This study shows that components of the oxidised lipid in atheroma, the hydroxyoctadecadienoic acids, may be involved in regulating disease progression by promoting development of macrophages with protective phenotype. Effects of the two HODEs were broadly similar in THP1 cells, but findings of this study were consistent with the notion that 13-HODE might exert protective effects in early atherosclerosis while the combination of 9-HODE and 13-HODE in later stages may promote atheroma formation. The receptors GPR120 and GPR132 were shown, for the first time, to be expressed on THP-1 cells using immunohistochemistry. In this work, we have examined the role of GPR132 in the control of inflammation mediated by HODEs. These oxidised lipids appear to regulate the differentiation of monocytes into a protective M2 phenotype. HODEs are abundant in atherosclerotic plaque, and have important regulatory functions. This work suggests that measurement of HODEs or expression of the receptors for long-chain fatty acids may be significant biomarkers for the atherosclerotic process. Perhaps more important, the effects of HODEs through cell surface G protein-coupled receptors and nuclear transcription factors may represent novel therapeutic targets leading to development of treatments that retard the process of atheroma formation.
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