Chapter 49

PPAR Nuclear Receptors and Altered RPE

Lipid Metabolism in Age-Related Macular

Degeneration

Goldis Malek, Peng Hu, Albert Wielgus, Mary Dwyer, and Scott Cousins

Abstract The pathophysiology of ‘early’ dry age-related macular degeneration (ARMD), characterized by the accumulation of lipid and protein-rich sub-retinal deposits remains largely unknown. Accumulation and dysregulated turnover of lipids as well as extracellular matrix (ECM) molecules in sub-retinal pigment epithelial (RPE) deposits and Bruch’s membrane, itself an ECM, play a role in ARMD. Epidemiological studies have shown an increased risk for the disease associated with higher dietary intake of long chain poly-unsaturated fatty acids (LCPUFA) and specifically more so for n-6 versus n-3 fatty acids. PUFAs are membrane targets of lipid peroxidation and natural ligands for the nuclear receptors, peroxisome proliferator activated receptors (PPAR). Here we investigated the expression of genes involved in lipid metabolism and expression of the three isoforms of PPARs in an immortalized cell line of human RPE cells (ARPE19) in the presence or absence of fatty acids.

49.1 Introduction

Age-related macular degeneration (ARMD) is the leading cause of visual impairment in the Western world. It is a late on-set progressive degeneration involving the photoreceptors, neurosensory retina, Bruch’s membrane, and the choriocapillaris. At the center of the disease is changes and degeneration of the retinal pigment epithelial cells (RPE). Clinically, ARMD progresses in different stages. Early or dry is characterized by the accumulation of lipid and protein rich extracellular deposits under the RPE including drusen, basal linear deposits and basal laminar deposits (Curcio and Millican 1999) collectively referred to as sub-RPE deposits. Geographic

G. Malek (B)

Department of Ophthalmology, Duke University, Durham, NC, USA e-mail: gmalek@duke.edu

Medicine and Biology 664, DOI 10.1007/978-1-4419-1399-9_49,C Springer Science+Business Media, LLC 2010

atrophy is characterized by RPE atrophy and late or wet/exudative ARMD is characterized by endothelial invasion and pathological neovascularization under the retina (Bird et al. 1995; Green 1999). Though treatment options for the wet form of the disease are currently available and to some extent successful, there hasn’t been any breakthrough in identification of drugs that directly target the sub-RPE deposit formation found in 85–90% of total ARMD patients living with this burden (Klein et al. 2007). Therefore, it is critical to further our understanding of the molecular and biological mechanisms that contribute to drusen formation.

49.1.1Current Hypotheses Surrounding Sub-RPE Deposit Formation

The pathogenesis of dry ARMD is still poorly understood. Various epidemiologic risk factors have generated specific hypotheses. These include: aging and RPE lysosomal failure evident by accumulation of metabolic waste and lipofuscin formation; family history/genetics including risk associations with polymorphisms of CFH, LOC387715/ARMS2, HrtA-1, APOE, VEGF, MMP-9, and the mitochondrial gene MTND2 LHON4917G (4917G) (Schmidt et al. 2002; Edwards et al. 2005; Fiotti et al. 2005; Hageman et al. 2005; Haines et al. 2005; Klein et al. 2005; Yang et al. 2006; Ross et al. 2007; Canter et al. 2008); chronic oxidative injury and inflammation for which both the retina and RPE are particularly vulnerable due to the high levels of cumulative irradiation they are exposed to overtime and the composition of long chain polyunsaturated fatty acids (PUFA) which can be easily oxidized; and, lipid dysregulation and accumulation in RPE cells, Bruch’s membrane and sub-RPE deposits. Modifiable risks associated with ARMD include smoking and diet. Though the stimulus for drusen formation is unknown, much of its content has been revealed by histochemistry, immunohistochemistry and proteomics (Crabb et al. 2002) to be composed of esterified and unesterified cholesterol, apolipoproteins (Malek et al. 2003; Curcio et al. 2005), vitronectin (Hageman et al. 1999), inflammatory proteins such as amyloid P, C5, CFH, C5b-9 (Hageman et al. 2001; Anderson et al. 2002; Johnson et al. 2002; Anderson et al. 2004), crystallins, calcium and many others.

49.1.2Long Chain Poly-Unsaturated Fatty Acids (LCPUFA) are Associated with ARMD Risk

Fatty acids are not only a source of energy but also essential to physiological cell functions working as modulators of cellular signaling and metabolism, signal transduction, cell growth, differentiation, and membrane lipid composition. The significance of PUFAs are highlighted by the findings that altered levels of PUFA and their metabolites are quite common in obesity, atherosclerosis and cancer (B.M Forman, PNAS, 1997). Humans lack the 15 and 12 desaturase enzymes to de novo synthesize essential fatty acids, which are particularly rich in PUFAs, therefore they

are dependent on dietary sources of these compounds (Sampath and Ntambi 2005). Depending on the position of the first double bond from the methyl end of the carbon chain, PUFAs are subdivided into n-6 (linoleic and arachidonic acids) and n-3 (linolenic, eicosapentanoic and docosahexanoic acid). Epidemiological studies have concluded that there is an inverse relationship between dietary intake of n-3 PUFA and risk of developing ARMD (SanGiovanni et al. 2007, 2008), a relationship also reported in atherosclerosis, cancer, Alzheimer’s disease and diabetes. It is important to note that the pro-inflammatory nature of PUFAs and their propensity to lipid peroxidation is determined by both the n-6/n-3 PUFA ratio and the total PUFA content of tissue. Within the cell, PUFAs are natural ligands for nuclear receptors called peroxisome proliferator activated receptors (PPARs).

49.1.3Peroxisome Proliferator Activated Receptors (PPARs) are Expressed in ARPE19 Cells

Peroxisome proliferator-activated receptors (PPARs), members of the steroid/thyroid nuclear receptor superfamily, are transcription factors activated by fatty acids and their derivatives. They are widely expressed and known to stimulate enlargement of peroxisomes, which contain a variety of oxidative metabolic processes such as beta-oxidation enzymes. After binding to endogenous ligands such as fatty acids/low density lipoproteins (LDL) or synthetic agonists, PPARs heterodimerize with another nuclear receptor, retinoid X receptor (RXR). This PPAR/RXR obligate heterodimer then binds to specific DNA response elements (PPRE) consisting of a direct repeat of the consensus hexameric motif AGGTCA interspaced by a single nucleotide, initiating DNA transcription and upregulation of specific PPAR genes. Functionally, they modulate the activity of genes involved in many processes including lipid homeostasis, glucose regulation, immune regulation, cell differentiation, inflammation, wound healing and have been associated with ischemia, cancer, systemic diseases such as atherosclerosis and diabetes, and age-related neurodegenerative diseases that share pathogenic mechanisms with ARMD including Alzheimer’s disease (Kersten et al. 2000). In fact, PPARs and their high affinity synthetic agonists (fibrates and TZDs) have been marketed for hypercholesterolemia and type 2 Diabetes mellitus (Kersten et al. 2000; Olefsky and Saltiel 2000).

At least three isoforms of PPARs have been identified, alpha-α, delta-δ (also known as beta or NUC1) and gamma-γ, with the subtypes overlapping in activity, function and location. Currently there is no consensus as to the overall combined function of PPARs. PPAR-α is highly expressed in the liver, heart, muscle and kidney, as well as in cells of the arterial wall, while PPAR-γ is expressed at high levels in white adipose tissue, where it activates adipocyte differentiation, as well as foam cells, activated macrophages that play a major role in the pathogenesis of atherosclerosis (Chawla et al. 2001; Chinetti et al. 2001; Moore et al. 2001). PPAR-δ is ubiquitously expressed in various tissues and is one of the key regulators of energy