Chapter 15

Deposition and Oxidation of Lipoproteins in Bruch’s Membrane and Choriocapillaris Are “Age-Related” Risk Factors with Implications in Age-Related Macular Degeneration

Ignacio R. Rodriguez

Abstract This chapter reviews the deposition and oxidation of LDL in Bruch’s membrane (BrM), the retinal pigment epithelium (RPE), and the choriocapillaris (CH) and the potential consequences of this deposition in relation to age-related macular degeneration (AMD). Lipid deposition and oxidation are age-related effects that when considered systemically play an important role in most if not all agerelated diseases. Oxidized lipid deposits are found throughout the vascular system where they can initiate chronic inflammatory responses that cause cellular stress and loss of function. Aging is a complex multifactorial process that sets the stage for genetic and environmental factors that initiate the pathogenesis of AMD and other age-related diseases.

15.1Introduction

A properly functioning retinal pigment epithelium (RPE) is essential to the health of the retina and especially to the viability of the photoreceptors [1]. The maintenance of the photoreceptors is a critical function of the RPE and the loss of this function is widely accepted as the central cause of age-related macular degeneration (AMD) [1, 2]. Aging and lipoprotein accumulation are suspected of causing RPE malfunction and oxidative stress [3, 4]. The retina vasculature may also play an important role in the development of AMD. The retina has two main vascular sources originating from branching of the ophthalmic artery; one branch becomes the central retinal artery which supplies the neural retina and another branch supplies the choriocapillaris (CH) [5]. Endothelial cells in the neural retinal capillaries contain

I.R. Rodriguez (*)

Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, Building 6, 136, 6 Center Dr, Bethesda, MD 20892, USA

e-mail: rodriguezir@nei.nih.gov

in Applied Basic Research and Clinical Practice, DOI 10.1007/978-1-61779-606-7_15, © Springer Science+Business Media, LLC 2012

tight junctions and are surrounded by Müller cells [5]. This gives increased protection to the neural retina, since oxygen, nutrients, and waste products must cross two cell layers. However, in the back of the retina the CH is fenestrated, thus leaving the RPE as the only barrier between the blood and the neural retina [5]. Bruch’s membrane lies between the choroid and RPE, but this is a basement membrane not designed to exclude molecules. The RPE is a multifunctional tissue that serves as a selective barrier through which nutrients, oxygen, and waste must pass on the way into and out of the retina. Hence, the RPE plays a major supporting role to the photoreceptors which are highly specialized cells with very high metabolic activity [6]. This makes the function of the RPE essential to the pathogenesis of AMD. Therefore, the deposition and oxidation of lipids in and around the RPE may be the most significant “age-related” risk factor in the pathogenesis of AMD.

15.2Aging

Aging is the most important risk factor in AMD [3] and therefore it needs to be in the forefront when considering factors that may affect its pathogenesis. Aging is the result of an imbalance between damage and cellular repair systems. It is a battle that we are all destined to lose. In humans, aging diseases have only become a major health issue in the last 200–300 years. Technological advances have increased human survival significantly and presently most people in developed countries are expected to live long enough to experience age-related diseases [7]. This is a relatively recent event, since much of human evolution was spent in small family groups or tribes where few individuals lived much beyond 45 or 50 years of age [7, 8]. Therefore natural selection only affected genes that determined fitness in the first 2–3 decades of life, when reproduction and survival of the next generation was important [7, 8]. Diseases such as atherosclerosis, cancer, type II diabetes, Alzheimer’s disease (AD), and AMD were an insignificant health issue during most of human evolution since few individuals lived long enough to experience them. There are many different theories regarding aging and these have been reviewed extensively elsewhere [7–14]. The general consensus is that aging is caused by gradual accumulation of cellular damage [11–14]. Inflammation and the involvement of NFkB [12] and its related pathways [13, 14] have been long suspected of being involved in aging. Our results suggest that inflammation and the NFkB pathways are involved in cellular responses to oxidized LDL and oxidized cholesterol (7-ketocholesterol) (7KCh) [4, 15]

The other two important risk factors in age-related diseases are genetics and environmental factors (Fig. 15.1). Most of the aging process is influenced by the inevitable oxidation–inflammation cycle which degrades cellular function. The response to this oxidation is likely mediated by primary genetic and environmental factors. The primary genetic factors are likely genes involved in the maintenance and repair of fundamental cellular systems [7–14]. Mutations in these genes are known to cause premature aging [7–14] but the effects of minor sequence variants

sanitation, and health care early in life [7, 8]. These primary environmental and genetic factors influence overall health and could also influence the way an individual ages later in life. Secondary factors come to play later, mostly in adulthood when the effects of aging are becoming more evident. Such secondary environmental factors generally can be under some form of control by the individual. For example, diet, exercise, smoking, and other habits can affect overall health. The secondary genetic factors are likely mild mutations or sequence variants that are either neutral or beneficial early in life and therefore not subjected to any form of natural selection. These secondary genetic variations may affect gene expression or may reduce gene function and only become problematic when other systems are also beginning to fail. Such genetic factors become important when the organism reaches a critical threshold period before the onset of the age-related diseases. There may also be some overlap between primary and secondary effects, making age-related diseases even more challenging to predict and comprehend.

15.3Deposition and Formation of Oxidized LDL

The deposition and formation of oxidized LDL is the central cause of atherosclerosis [16, 17]. LDL is a complex lipid particle with the largest known protein at its core (apoB-100, ~540 kDa) [18]. LDL contains cholesterol, cholesterol-fatty acid esters, various triglycerides, and phospholipids [18]. Lipids compose approximately 80% of the dry weight of the particle and proteins (mostly apoB) the remaining 20% [18]. Approximately 60% of the lipid portion is composed of free cholesterol and cholesteryl-esters [18]. LDL seems to transiently coat arteries and capillaries but in some vascular areas LDL can form more permanent deposits [16–20]. This may be due to changes in the membranes of the endothelial and/or vascular smooth muscle cells, perhaps due to some form of damage to the vascular system (e.g., high blood pressure). This deposition seems to be counteracted by immune cells, especially macrophages, which readily internalize LDL deposits [16–20]. These deposits, once formed, are highly susceptible to autooxidation [16–20] by catalytic levels of Cu+2 and Fe+2 [21, 22]. Polyunsaturated fatty acids (PUFAs) which are commonly found esterified to cholesterol in LDL will also oxidize at their respective double bonds [22]. This complex mixture of oxidized lipids is known to have serious detrimental consequences to the macrophages that phagocytize them [16–20]. During the aging process, perhaps due to loss of macrophage function or perhaps to a reduced number of scavenging cells, these deposits oxidize to highly toxic levels [16–22]. The oxLDL-laden macrophages are known as “foam cells” which are a major component of atherosclerotic plaques [16–20].

In the retina, this process of LDL deposition and oxidation is also occurring but its consequences are not as well understood [3, 4]. The accumulation of lipids as a consequence of aging has been well characterized by Curcio and her colleagues [3, 23]. Our group has also shown that apoB containing deposits can form throughout the CH and BrM in rats injected with human LDL [24, 25]. Moreover, using an

Fig. 15.2 Aging in the RPE-BrM-Choroid. The first 3–4 decades are generally uneventful with only the gradual increase in lipofuscin granules as the most evident of intracellular changes. The fourth and fifth decades are the beginning of the “critical threshold period” when lipid accumulation and oxidation in BrM and choriocapillaris initiate chronic inflammation. This is the time when secondary environmental and genetic factors may come into play (Fig. 15.1). Lipofuscin accumulation increases and the inflow of oxygen and nutrients and the outflow of photoreceptor waste and metabolites are restricted by the accumulated lipids. These accumulated lipids are increasingly oxidized causing a chronic inflammatory response in the RPE and choroidal endothelial cells. Oxidized lipids also attract microglia which may intervene in a positive or negative manner depending on the secondary factors. The combination of secondary environmental and genetic factors will influence how the disease progresses. Choroidal vessels may break out though BrM and cause the “wet” form of the disease or the RPE may continue to slowly degrade leading to “dry” orgeographic atrophy

antibody to oxidized cholesterol, 7-ketocholesterol (7KCh), our group also detected oxidized lipoprotein deposits throughout the RPE, BrM, and the CH in young monkeys [26]. 7KCh was also detected and quantified by high-performance liquid chro- matography-mass spectrometry (HPLC-MS) demonstrating unequivocally the oxidized nature of these deposits [26].

Figure 15.2 demonstrates a hypothetical aging process of the CH/RPE/photoreceptor area of the retina. Fresh lipids (mostly LDL) enter the retina by crossing through BrM into the RPE as previously proposed [27]. The RPE expresses the LDL receptor thereby attracting LDL into this area [24, 27]. Lipids from LDL particles are then processed by the RPE and transported to photoreceptors and other cells. The retina and RPE use the same proteins that the systemic “reverse cholesterol”

pathway uses [25, 27]. As the individual ages, BrM thickens due to lipid accumulation [3, 23] until it reaches a critical threshold level (Fig. 15.2). This point is defined by the initiation and progressive loss of RPE function. While the loss of RPE function is assumed to be gradual, a critical threshold period is reached when RPEdependent functions begin to fail. At this point the RPE is unable to internalize sufficient amounts of fresh lipids and is also unable to properly eliminate oxidized lipids and other waste products phagocytized from photoreceptors. Lipofuscin accumulation accelerates and the granules start to fuse with other vacuoles increasing the potential for oxidation (photooxidation) and the generation of toxic lipids. Although A2E, the main component of lipofuscin, is a poor photosensitizer [28, 29], it can photooxidize lipids if mixed at sufficiently high concentrations (unpublished results). This likely causes stress responses in the RPE which may respond by plasma membrane “blebbing” to expel unprocessed cellular debris toward BrM (Fig. 15.2). We have observed cultured RPE cell blebbing when stressed by internalization of oxLDL (unpublished data). This blebbing process may be the beginning of drusen formation. The formation of drusen deposits is considered a major risk factor for developing AMD [1, 2]. At some point during this process microglia and other immune cells become involved [30–32]. These cells may be attracted to the area either by cytokines released by the RPE or by encountering the oxidized lipid deposits during their normal migration. Microglia seem to be activated by oxidized lipids [33] which are abundant in BrM [23]. One interesting effect of microglia is that it can alter the function of the RPE [30, 32]. The role of microglia is under intense investigation and at this time is unclear whether they play a positive or a negative role [32, 33]. In this author’s opinion they seem to do both, depending on the timing and the particular genetic/environmental conditions of the individual. Oxidized LDL and one of its main component, 7KCh, can cause inflammatory responses in the RPE [4, 15, 26] and are likely to elicit similar responses from microglia. This chronic inflammatory process likely begins before the critical threshold period and culminates during this time to cause major functional deficiencies in the RPE (Fig. 15.2). At the later stages of this critical threshold period the CH may also respond to an increase in VEGF release. In cases where BrM is compromised, choroidal vessels may proliferate into the neural retina causing “wet” or exudative AMD (Fig. 15.2). In other cases the RPE may gradually degenerate leading to photoreceptor and eventually to RPE death (“dry” or atrophic AMD and geographic atrophy).

15.4Inflammatory Consequences of Oxidized LDL Deposition

The main consequences of oxidized LDL deposition are inflammation and cytotoxicity [4]. These are related processes that seem to be cell-type and dose dependent [4, 15]. The most problematic component in oxLDL is 7KCh [15, 26, 34] which can be found both free and esterified to various fatty acids [22]. In atheromatous plaques 7KCh concentrations has been measured at levels in excess of 100 mM [35].

Fig. 15.3 Inflammatory pathways initiated by oxLDL and 7KCh. The pathways most likely involved in the chronic inflammatory response caused by oxLDL and 7KCh in RPE and choroidal endothelial cells are shown. The precise mechanism of how such pathways are triggered is unclear. The PLA2/ACAT may be involved since we and others have observed the formation of 7KFAEs. The relationship between the 7KFAEs and the NFkB inflammatory pathways is under investigation

In cultured RPE cells, 7KCh can cause significant inflammatory responses in low micromolar concentrations and is lethal at concentrations above 10 mM [15].

7KCh-induced inflammation is a complex process that is not fully understood [4, 15, 26, 34] (Fig. 15.3). Various groups have investigated this process and have published different and sometimes conflicting results [4]. However, there seems to be a consensus regarding the involvement of the NFkB and MAPK pathways [4, 12, 15, 36–39], but the mode of activation is not well understood. Some groups suggest that NFkB activation occurs via reactive oxygen species (ROS) [40–44]. In our hands, 7KCh did not induce NOX-4 or ROS in four different cell types tested [15]. The reason(s) for these discrepancies remains unknown but we suspect the method of delivery and the high concentrations of 7KCh used by other groups (~100 mM) may be responsible. We typically deliver 7KCh complexed with hydroxypropyl-b- cyclodextrin (HPBCD) dissolved in PBS [15, 26]. We analyze our stock solutions to

ensure the concentration of 7KCh does not change through the filter-sterilization process. We add enough additional HPBCD (30-fold molar excess) to maintain 7KCh solubility during subsequent dilutions from stocks to working solutions and to the cell media. Using this method, inflammatory and cytotoxic reactions can be achieved at concentrations between 8 and 15 mM 7KCh without carrier effects [15, 26]. Other groups [40–44] use ethanol or DMSO to deliver 7KCh and may be prediluting the 7KCh in their media. 7KCh is very adherent to glass and plastic surfaces, and this can cause great variability in the concentrations actually delivered to cells. This is the reason we believe that other groups must use concentrations of 7KCh in excess of 100 mM to demonstrate toxicity. At these high concentrations, cells die quickly and carriers such as ethanol can generate ROS independently.

Using a variety of specific inhibitors we have determined that 7KCh activates NFkB mainly via PI3K/AKT/NFkB or PI3K/PKCz/NFkB [15] (Fig. 15.3). Our data indicates that MAPKs (p38 and ERK) are involved in cytokine induction but not directly in NFkB activation, possibly by activating other transcription factors that work in combination with NFkB [15]. However, new evidence (unpublished data) suggests that MAPKs may activate NFkB if the upstream PI3K is completely inhibited, thus allowing an attenuated but measurable cytokine induction (Fig. 15.3). Therefore, such “cross talk” between these pathways seems to allow some measurable activation of NFkB under a variety of conditions.

Several studies suggested that phospholipase A2 (PLA2) activation by oxLDL is involved in apoptotic signaling in macrophages [45–48]. These studies suggest that PLA2 translocates from the cytosol to the plasma membrane in response to oxLDL and 7KCh [46–48]. One study suggested that PLA2 releases arachidonic acid (20:4) which is then sterified to 7KCh by acyl-coenzyme A: cholesterol acyltransferase (ACAT) [48]. The 7KCh-20:4 ester is suspected of causing apoptosis in macrophagederived PD388D1 cells [48]. Studies in our laboratory indicate that treatment of cultured RPE cells with 7KCh cause the formation of various 7KCh-fatty acid esters (7KFAEs) at the onset of cellular toxicity (unpublished results). Analyses of extracts from the 7KCh-treated cells by HPLC-MS have identified at least ten different types of 7KFAEs with fatty acid moieties ranging from C16 to C28. However, the majority of the 7KFAEs seem to be in three mass peaks with parental ions suggesting fatty acids 18:3, 20:4, and 20:3 (Fig. 15.4).The effects of these 7KFAEs on the activation of the 7K-mediated inflammatory pathways is under investigation.

One important piece of evidence that is helping to connect 7KFAEs to the inflammatory process is the inhibition of 7KCh-mediated inflammation by certain types of PUFAs. The inhibitory effect of PUFAs on cholesterol esterification has been known for many years [49, 50]. Docosahexanoic acid (DHA), a well-known essential fatty acid, was recently found to inhibit mitochondrial acetyl-CoA acetyltransferase (ACAT1) [51]. Moreover, the protective effects of PUFAs, especially Omega-3 fatty acids, had been demonstrated both experimentally and clinically [52, 53]. However, the mechanism(s) by which PUFAs exhibit these beneficial properties is not well understood. We have found that several PUFAs, including DHA, are antagonistic to 7KCh-mediated inflammation (Fig. 15.4). In cultured RPE cells, PUFAs such as