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Keywords:

  • drusen;
  • lipofuscin;
  • macular degeneration;
  • retinal pigment epithelium

Abstract.

  1. Top of page
  2. Abstract.
  3. Introduction
  4. RPE in the Pathogenesis of AMD
  5. Protein Degradation Processes in RPE
  6. Conclusion
  7. References

Age-related macular degeneration (AMD) is attributed to a complex interaction of genetic and environmental factors. It is characterized by degeneration involving the retinal photoreceptors, retinal pigment epithelium (RPE) and Bruch’s membrane, as well as alterations in choroidal capillaries. AMD pathogenesis is strongly associated with chronic oxidative stress and inflammation that ultimately lead to protein damage, aggregation and degeneration of RPE. Specific degenerative findings for AMD are accumulation of intracellular lysosomal lipofuscin and extracellular drusens. In this review, we discuss thoroughly RPE-derived mechanisms in AMD pathology.


Introduction

  1. Top of page
  2. Abstract.
  3. Introduction
  4. RPE in the Pathogenesis of AMD
  5. Protein Degradation Processes in RPE
  6. Conclusion
  7. References

Risk factors of AMD

Age, smoking, family history, gender, high blood pressure, hypercholesterolaemia and arteriosclerosis have been shown to be the most important risk factors for the development of age-related macular degeneration (AMD) – the most common cause of central vision defects in the elderly in the Western countries (Klein et al. 1998; Age-Related Eye Disease Study Research Group 2000; Smith et al. 2001). A strong genetic predisposition, which most commonly localizes 1q25-31 and 10q26 chromosomes, has been linked to AMD. The first associations observed were between the Y402H polymorphism (rs1061170) of the complement factor H gene and AMD in several populations (Edwards et al. 2005; Hageman et al. 2005; Haines et al. 2005; Klein et al. 2005; Souied et al. 2005; Lau et al. 2006; Seitsonen et al. 2006). Later, an association between the LOC387715/HTRA1 locus and AMD in populations of different origin has been documented (Rivera et al. 2005; Dewan et al. 2006; Yang et al. 2006; Lu et al. 2007; Mori et al. 2007; Ross et al. 2007; Tanimoto et al. 2007; Weger et al. 2007; Yoshida et al. 2007). Before the finding of the polymorphisms in the complement system genes, apolipoprotein E (APOE) allelic polymorphism was implicated in AMD. Individuals carrying the APOE-e4 allele seemed to have a lower risk for the disease, whereas the APOE-e2 allele was linked to an elevated risk (Klaver et al. 1998; Schmidt et al. 2002; Zareparsi et al. 2004; Baird et al. 2006).

Classification and phenotypes of AMD

Clinically, AMD can be classified into early- or late-atrophic AMD (dry AMD) and exudative (wet AMD) (Jager et al. 2008). In atrophic AMD, retinal pigment epithelium (RPE) and photoreceptors in the macular area gradually degenerate leading to central visual loss (Ambati et al. 2003). In association with cellular degeneration, the pathogenesis of AMD also involves the accumulation of extracellular deposits, called drusen, between the RPE and Bruch’s membrane (Anderson et al. 2002; Jager et al. 2008). Drusen are classified morphologically either as hard or soft deposits. Hard drusen are yellow-white lesions, typically <63 μm in diameter, whereas soft drusen are defined as being larger than 63 μm in diameter and usually appear during the late stage of the disease (Ambati et al. 2003). Soft drusen is estimated to become confluent, and multiple soft drusens are an independent risk factor for visual loss in AMD (Macular Photocoagulation Study Group 1997). Detailed cellular mechanisms of drusen formation is still a mystery, even though immunohistochemical analyses indicate accumulation of many inflammation-related proteins such as apolipoproteins B and E, different immunoglobulins, factor X, amyloid P component, complement C5 and C5b-9 terminal complexes, fibrinogen and vitronectin in drusens (Hageman & Mullins 1999; Anderson et al. 2001). A prospective evaluation of patients with drusen in the fellow eyes of unilateral exudative AMD suggested that the risk of developing exudative AMD in the second eye peaks at 4 years, with an increasing incidence of geographic atrophy thereafter (Sarraf et al. 1999). Increased accumulation of drusens may trigger hypoxia in RPE cells and accelerate RPE degeneration that secondarily evokes cell death in neural retina (Dunaief et al. 2002; Arjamaa et al. 2009; Stefansson et al. 2011).

Relatively rapid visual loss in patients with AMD is mostly caused by exudative AMD (Bressler et al. 1988). It is characterized by proliferation of choroideal neovascularization (CNV) that is a clinical hallmark of exudative AMD. Excessive expression of vascular endothelial growth factor (VEGF) by RPE cells leads to new vessel formation originating from the choroid and extending through defects in the Bruch’s membrane and the RPE sub- and intraretinally. Choroideal neovascularization arise as capillary-like structures with multiple points of origin (Green & Enger 2005; Green & Key 2005). Choroideal neovascularization usually causes serous detachment of the RPE or retina, RPE tears, haemorrhages and lipid exudation. Vision loss occurs through the structural and metabolic damages caused by exudates and haemorrhages, and the secondary reactive gliosis and cell death (Ambati et al. 2003).

RPE in the Pathogenesis of AMD

  1. Top of page
  2. Abstract.
  3. Introduction
  4. RPE in the Pathogenesis of AMD
  5. Protein Degradation Processes in RPE
  6. Conclusion
  7. References

Oxidative stress triggers detrimental effects on RPE

The most outer segment of the retina consists of photoreceptors that are metabolically supplied by RPE cells. The latter serve a variety of metabolic and supportive functions that are of vital importance for retinal photoreceptors, including maintenance of the blood–retina barrier, regulation of ion balance, participation in the visual cycle, express various growth factors and phagocytic uptake and degradation of constantly shed apical photoreceptor outer segments (POS) (Kim et al. 1999; Strauss 2005; Barnstable & Tombran-Tink 2004; Filleur et al. 2009). Photoreceptor outer segments phagocytosis is a central homeostatic cellular process in RPR cells. Retinal pigment epithelium membrane receptors, CD36, integrin αvβ5 receptor and the mannose receptor have been shown to mediate phagocytosis of POS (Boyle et al. 1991; Ryeom et al. 1996). The secreted glycoprotein milk fat globule-EGF 8 (MFG-E8) has shown to promote the synchronized clearance of POS by the RPE by acting as a ligand for αvβ5 integrin receptors (Nandrot et al. 2007).

Oxidative stress refers to a progressive cellular damage caused by reactive oxygen species (ROS) contributing to protein misfolding and evoking functional abnormalities during RPE cellular senescence. It has a significant role in the pathogenesis of AMD (Beatty et al. 2000). The phagocytosis of POS by RPE cells generates oxidative stress caused by ROS (Tate et al. 1995). In addition high oxygen consumption and long periods of exposure to light evoke oxidative stress to RPE cells (Beatty et al. 2000). One explanation of the RPE degeneration is an age-related phagocytic, protein degradation and metabolic insufficiency. This is characterized by progressive accumulation of lipofuscin in lysosomes and decreased cellular capacity to degrade POS material or cytoplasmic proteins in lysosomes (Wing et al. 1978; Delori et al. 2001; Algvere & Seregard 2002; Sparrow & Boulton 2005; Warburton et al. 2005; Krohne et al. 2010). Retinal pigment epithelium lipofuscin is a photoinducible generator of ROS that disturbs lysosomal integrity, induces lipid peroxidation, reduces phagocytic capacity and causes RPE cell death (Fig. 1; Wassell et al. 1999; Wassell et al. 1999; Sundelin et al. 1998; Boulton et al. 1993; Holz et al. 1999). Intracellular, autofluorescent and auto-oxidant lipofuscin contains vitamin A-derived fluorophores that inhibit mitochondrial respiration and promote protein misfolding (Pauleikhoff et al. 1990; Sparrow et al. 2003; Terman & Brunk 2004; Kaarniranta et al. 2005; Nordgaard et al. 2008). Moreover, impaired lysosomal degradation results in a significant reduction in autophagy, another clearance mechanism in RPE cells (see later, Krohne et al. 2010). Increased lipofuscin accumulation in RPE cells is observed in vivo by fundus autofluorescence imaging and has been demonstrated to precede RPE cell damage and AMD advancement (Bindewald et al. 2005; Holz et al. 2007).

image

Figure 1.  Schematic representation of protein aggregation in aged retinal pigment epithelial cells (RPE). Retinal pigment epithelium cells digest retinal outersegment discs that are endocytosed and fused with lysosomes to be degraded. In aged RPE cells, lysosomal degradation is impaired resulting in accumulation of lipofuscin that is auto-oxidant material increasing oxidative stress and protein damage in the RPE cells. Heat-shock proteins attempt to repair formed protein damages, but this process is estimated to be disturbed in aged cells. Simultaneously, proteasomal and autophagy protein clearance systems are not working as effectively as in young RPE cells. Thus, the efficiency of central proteolytic machines is impaired in aged cells; proteins are apparently moved via exocytosis to the outside of the RPE cells. This material might be involved in drusen formation together with chronic inflammation and inflammatory cells.

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In addition to intracellular lipofuscin accumulation, extracellular drusens contain advanced glycation end-products (AGE), high level of oxidized low-density lipoproteins (oxLDL) and oxysterols (Ishibashi et al. 1998; Crabb et al. 2002; Javitt & Javitt 2009). The receptor for AGE (RAGE) is a multi-ligand receptor recognising modified proteins (Bierhaus et al. 2005) normally not markedly expressed in the retina, but highly accumulated in RPE cells, photoreceptors and choriocapillaris in advanced AMD (Yamada et al. 2006; Howes et al. 2004). The nuclear factor kappaB (NFκB) system is a master regulator of both innate and adaptive immunity that is activated with RAGE. The increased AGE and RAGE strongly activates NFκB also in cultured RPE cells and evokes apoptotic cell death (Howes et al. 2004). Furthermore, NFκB is a central regulator of IL-6, a cytokine that has been shown to be an important regulator of CNV (Izumi-Nagai et al. 2007; Koto et al. 2007; Paimela et al. 2007). The activation of RAGE induces the expression and secretion of VEGF in RPE cells, and hence the activation of AGE–RAGE axis can evoke CNV, similar to the induction by oxLDL and oxysterols (Oskolkova et al. 2008; Smith et al. 2001).

Protein Degradation Processes in RPE

  1. Top of page
  2. Abstract.
  3. Introduction
  4. RPE in the Pathogenesis of AMD
  5. Protein Degradation Processes in RPE
  6. Conclusion
  7. References

Heat-shock proteins, proteasomes and autophagy regulate clearance in RPE

Similar to other cell types, RPE survival is dependent on efficient removal of misfolded proteins from the cytoplasm. Harmful misfolded protein aggregates can be destroyed by several mechanisms. First, heat-shock proteins (HSPs) are known to function as molecular chaperones that participate in protein damage-repair process and prevent harmful protein aggregation in cells (Hartl 1996; Hartl & Hayer-Hartl 2002). If this process is not successful, potentially toxic proteins are targeted to the proteasome machinery for degradation. Misfolded proteins are tagged with a small polypeptide ubiquitin that directs the complex to the ubiquitin/proteasomal protein degradation pathway. Dysfunction of this ubiquitin-proteasome system might cause aggregation of damaged proteins into larger bodies called aggresomes (Fig. 1; Kopito 2000). It is believed that up to 80–90% of all intracellular proteins, especially oxidative stress damaged proteins, are degraded by the ubiquitin–proteasome proteolytic machinery. Lysosomal degradation including autophagy can clean endocyted proteins, membrane proteins and whole cell organelles (Epstein et al. 1996; Hershko & Ciechanover 1998; Ciechanover 2005). Recent findings reveal that there is a cross-talk between Hsps, proteasomes and autophagy in the regulation of protein aggregation in RPE cells (Ryhanen et al. 2009; Kaarniranta et al. 2010).

Heat-shock proteins repair misfolded proteins

Heat-shock proteins prevent the accumulation of cellular cytotoxic protein aggregates and assist in correct folding of dysfunctional and misfolded proteins prior to their translocation across the lysosomal membrane (Dice 2007). The HSPs are divided into different families (Hsp90, Hsp70, Hsp60, Hsp40 and small Hsps) according to their function, molecular size and cellular localization (Craig et al. 1994). HSP90 is an abundant cytosolic protein in mammalian cells comprising 1–2% of total soluble proteins and acting as a key component in the regulation of autophagy (see later, Buchner 1996). Furthermore, Hsp90 inhibition has shown to inhibit protein aggregation in the RPE cells (Johnston et al. 1998, 2002; Garcia-Mata et al. 1999; Mimnaugh et al. 2004; Ryhanen et al. 2011). The expression of these HSPs are increased in stress conditions involving protein damage, e.g. heat, hydrostatic pressure, ischaemia-reperfusion and oxidative stress (Kaarniranta et al. 1998, 2009). Interestingly, increased accumulation of Hsp90 and HSP27 has been observed in drusens (Fig. 1; Decanini et al. 2007), and Hsp70 are present in lysosomal fractions of RPE cells (Ryhanen et al. 2009). Therefore, many Hsps might have a regulatory role in repairing misfolded proteins and in lysosomal proteolysis in RPE cells.

Proteasomal protein degradation in RPE cells

The RPE cells live under chronic oxidative stress because of normal visual cycle metabolism and constant light exposure. During the ageing process, capacity to repair oxidative stress-induced cellular damage is decreased, which associates with impaired HSP response. This may trigger increased mass of misfolding proteins that have a tendency to gather into detrimental aggregates (Kaarniranta et al. 2009). If the HSP-linked protein folding fails, the misfolded proteins are tagged with ubiquitin and transferred to proteasomes for degradation (Kopito 2000; Wojcik 2002). Ubiquitin functions like a ‘stamp’ and directs the complex to the ubiquitin/proteasomal protein degradation pathway. The eukaryotic proteasome is a multicatalytic proteolytic complex that recognizes and selectively degrades oxidatively damaged and ubiquitinated proteins. However, there is a hypothesis that the accumulation of oxidized and ubiquinated proteins is because of the decrease in proteasome activity with age; alternatively, accumulation of cross-linked proteins with ageing can block or overwhelm the proteasomal system (Fig. 1). Under certain conditions, oxidative stress itself may inactivate the function of proteasomes and upregulate the release of proinflammatory cytokines (Kaarniranta & Salminen 2009; Kaarniranta et al. 2009), which may account for the chronic inflammation in retina and the accumulation of drusens, evoking development of AMD (Kaarniranta & Salminen 2009).

Autophagy

Autophagocytosis, a specific lysosomal clearance system, contributes to turnover of aggregate-prone proteins that is extremely important in postmitotic RPE cells. Autophagy is an intracellular process activated by stress and involved in protein and organelle degradation via the lysosomal pathway. It is involved in several age-related and inflammatory diseases (Levine & Kroemer 2008; Kaarniranta et al. 2009, 2011; Ryhanen et al. 2009). Autophagy is also involved in the host defence system linking it with the innate and adaptive immunities (Schmid & Münz 2007; Fesus et al. 2010).

Autophagy is categorized into different clearance systems. Macroautophagy begins with the formation of the autophagosome, a double-membrane-bound vesicle that contains larger cytoplasm deposits and/or cellular organelles. After their formation, autophagosomes undergo a maturation process including fusion events with endosomes and lysosomes for degradation (Figs 1 and 2A; Eskelinen & Saftig 2009). Microautophagy involves lysosomes invaginating and directly sequestering a portion of cytoplasm. Chaperone-mediated autophagy (CMA) is the most studied autophagy process. In CMA, lysosome-associated membrane protein 2A (LAMP-2A) and HSPs (HSP90, HSC70 and HSP40) bind the target proteins and direct them for transport across the lysosomal membrane (Cuervo & Dice 1996).

image

Figure 2.  (A) Transmission electron micrograph of the typical autophagy process in retinal pigment epithelium (RPE) cells. Engulfed materials are under digestion inside the capsule-like autophagolysomes (arrow). Scale bar 5 μm. (B) Scanning electron micrograph of RPE cells in culture conditions undergoing anoikis on a nonattaching poly(2-hydroxyethyl methacrylate) surface. The cells arrange in rosettes around a ‘ghost’ or dead RPE cell (in the centre).

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The formation of the autophagosome is partially regulated by AuTophaGy (ATG) proteins (Yorimitsu & Klionsky 2005; Xie & Klionsky 2007). mTOR is a serine/threonine protein kinase integrating the upstream signalling of ATG proteins to different downstream effectors, for example, to the ATG1 kinase in autophagosomal vesicle formation (Yorimitsu & Klionsky 2005; Pattingre et al. 2008). Rapamycin is a well-known inhibitor of mTOR function and a potent inducer of macroautophagy that reflects to prolonged lifespan. The mTOR complex can inhibit autophagy, whereas the signals inhibiting mTOR stimulate autophagic degradation. Stress-induced signals in the endoplasmic reticulum (ER) can also activate the autophagy (Yorimitsu et al. 2006; Salminen et al. 2010). Another regulator of the autophagosome formation is Beclin 1 (Xie & Klionsky 2007; Pattingre et al. 2008). In addition, one of the main proteins proposed to be involved in the uptake of protein aggregates is p62 (Bjorkoy et al. 2006). The p62 or sequestosome 1 (SQSTM1) is a multifunctional protein adapter that has many roles in cell signalling, transcription regulation, receptor internalization and protein turnover. The p62 was first characterized in polyubiquitinated protein aggregates in response to proteasomal inhibition (Kuusisto et al. 2001). p62 is one of the most important molecules regulating the packing and transporting of ubiquitinated, misfolded and aggregated proteins for clearance via autophagy in mammalian cells (Fig. 1; Kim et al. 2008; Kirkin et al. 2009). The ubiquitin-associated domain (UBA) of p62 enables noncovalent binding to ubiquitin or ubiquitinated substrate proteins that are targeted to proteasomal degradation, or alternatively ubiquitinated complexes are shuttled to lysosomal system for degradation (Seibenhener et al. 2004, 2007; Kim et al. 2008). Furthermore, p62 seems to play a central role in improving cellular viability when proteasomal pathways are disturbed in RPE cells (Viiri et al. 2010). It has been shown that p62 is expressed in many age-related diseases (Kuusisto et al. 2001, 2002; Zatloukal et al. 2002; Nagaoka et al. 2004; Mathew et al. 2009).

The role of proteasomes and autophagy in RPE cells and in AMD are under active studies. It has been shown that proteasomes and autophagy are activated during AMD-associated stress conditions such as caloric restriction, hypoxia, oxidative stress or inflammation (Arjamaa et al. 2009; Kaarniranta et al. 2009; Stefansson et al. 2011). There is also evidence that enhanced autophagy reduces the toxicity of the protein aggregates that accumulate in many other age-associated diseases (Salminen & Kaarniranta 2009). An effective autophagic clearance system has recently been documented also in human RPE cells. Furthermore, recent studies show that disturbed autophagy is involved in AMD pathogenesis (Ryhanen et al. 2009; Wang et al. 2009). It has been demonstrated that in human AMD donor samples, autophagic markers are accumulated and lysosomal activity is decreased. If lysosomal function is suppressed, as occurs during lipofuscin load, autophagic clearance is not functional leading to increased RPE cell damage (Kaarniranta 2010). Preservation of the autophagic activity is associated with a lower intracellular accumulation of damaged proteins, better ability to handle protein damage, improved RPE cell function and retarded disease progression (Ryhanen et al. 2009; Salminen & Kaarniranta 2009; Wang et al. 2009).

Innate immunity response in RPE

The innate immune system consists of specific cells and mechanisms that recognize and respond to pathogens in a generic way via pattern recognition receptors (PRRs). These receptors sense both pathogen-associated molecular patterns (PAMPs) displayed by micro-organisms and endogenous danger signals released by injured cells, i.e. danger-associated molecular structures (DAMPs) (Bianchi 2007). The innate immune system does not just detect the invading pathogens but also recognizes the damage caused by the pathogens – both PAMPs and DAMPs can trigger subsequent inflammatory responses. The PRR system can respond to the ligands in two ways: (i) they can evoke the phagocytosis and disposal of target structure or (ii) they can trigger signalling pathways that induce the expression of inflammatory mediators (Kaarniranta & Salminen 2009). The PRR systems consist of membrane-bound Toll-like receptors (TLRs) and cytosolic NOD-like receptors (NLRs) that are capable of forming large cytoplasmic complexes – inflammasomes. Toll-like receptors are mainly responsible for recognising breakdown products of the extracellular matrix (e.g. elastin, hyaluronic acid and fibronectin) and secreted HSPs; the NLRs and inflammasomes especially sense danger signals, such as oxygen radicals, ultraviolet B (UVB) and potassium (K+) efflux evoking inflammation-mediated self-defence mechanisms (Bianchi 2007; Miyake 2007; Pétrilli et al. 2007).

The activation of TLRs with specific ligands induces the expression and secretion of pro-inflammatory cytokines (e.g. IL-6, IL-8), angiogenetic chemokines (e.g. CXCL8, CCL2) and adhesion markers (e.g. ICAM-1, VCAM-1) (Elner et al. 2003, 2005; Kindzelskii et al. 2004; Kumar et al. 2004; Ebihara et al. 2007; Paimela et al. 2007). Subsequently, the cytokines can evoke the production of ROS and induce oxidative stress in RPE (Holtkamp et al. 2001; Yang et al. 2007).

The maturation and secretion of IL-1β and IL-18 cytokines are the hallmarks of NLR and inflammasomal activity (Salminen & Kaarniranta 2009). Direct ligands activating NLRs are still largely unknown. One hypothesis is that oxidative stress and lysosomal damage might activate NLRs. Most recently, autophagic dying cells have been shown to activate the inflammasome via NALP3, stimulating secretion of IL-1β and pro-inflammatory cytokines (Petrovski et al. 2011). This could also be relevant in advanced AMD where heavy RPE autophagy is a cause of cell death.

Retinal pigment epithelium cells have the main role in the immune defence in the retina. The development of AMD involves several insults, which most likely trigger danger signals and activate PRRs in the RPE layer (Hageman et al. 2001; Lotery & Trump 2007). Lipofuscin and A2-E in particular impair lysosomal function and can lead to lysosomal damage and PRR activation via cathepsin B (Bergmann et al. 2004; Sparrow & Boulton 2005; Halle et al. 2008). Retinal pigment epithelium cells transport nutrients and ions between the choriocapillaries and the photoreceptors, and during stress, these cells are exposed to ionic changes that may activate inflammasomes through K+ efflux (Strauss 2005; Pétrilli et al. 2007). Moreover, the accumulation of drusen under the Bruch’s membrane simultaneously with the morphological changes in Bruch’s membrane itself provides a milieu for RPE–immune system interactions (Sivaprasad et al. 2005; Kaarniranta & Salminen 2009).

It has been postulated that choroideal dendritic cells (DCs) are also activated and recruited by injured RPE and oxidized proteins and lipids in the Bruch’s membrane (Fig. 1; Hageman et al. 2001). The RPE cells respond to dendritic cell activation by secreting immune response modulators including vitronectin and apolipoprotein E (Johnson et al. 2001). Activation of choroidal DCs might also initiate an autoimmune response to retinal or RPE antigens (Hageman et al. 2001). Antiretinal and anti-RPE antibodies have been detected in the serum of patients with AMD (Niederkorn 1990; Penfold et al. 1990; Gurne et al. 1991). Inflammatory cells like multinucleated giant cells and leucocytes are involved in the later stages of AMD (Penfold et al. 1985; Killingsworth et al. 1990; Seregard et al. 1994). Activated macrophages and other inflammatory cells secrete enzymes that can damage cells and degrade the Bruch’s membrane. By releasing cytokines, inflammatory cells might promote the growth of CNV into the sub-RPE space (Fig. 1; Oh et al. 1999).

Clearance processes within and beyond the RPE

The early stages of atrophic form of AMD involve RPE degeneration and cell death via different cell death modalities (Hafezi et al. 1997; Valamanesh et al. 2007; Ryhanen et al. 2009). Formation of intraretinal cysts or pigment epithelial detachments (PED) as part of atrophic (intact Bruch’s membrane) and exudative (neovascular, penetrated Bruch’s membrane) type of AMD can be a contributing factor towards RPE death. Drusen accumulation and PED formation resemble a cell death phenomenon in RPE cells called anoikis that is caused by cell detachment from the extracellular matrix (ECM) (Gilmore 2005; Petrovski et al. 2011). During anoikis, cells try to survive the detachment by using the already dying or dead (ghost) cell membranes for attachment (Fig. 2B).

Intracellular lysosomal lipofuscin accumulation, on the other hand, resembles failure of either the proteasomal or the autophagosomal clearance system to cope with the overwhelming cellular debris, which under extreme conditions can also lead to cell death. Ultimately, failure to clear dead cells or debris may facilitate further drusen formation and progression of AMD (Forrester 2003).

The clearance processes in the retina can be described as nonprofessional (as in atrophic AMD), where healthy, nondedicated RPE cells engulf dying neighbouring cells, while the blood–retina barrier is intact; consequently, involvement of vascular-borne professional phagocytes (macrophages and DCs) develops in the exudative form of AMD, the price of which is accumulation of chemoattractant material and low-grade inflammation (Forrester 2003; Irschick et al. 2004; Petrovski et al. 2011).

Dying cells present on their surface different ‘eat-me’ signals that get recognized by the engulfing cells in a so-called third synapse (Majai et al. 2006). New eat-me signals are continuously discovered. Recently, an open reading frame phage display discovered Tubby and Tubby-like protein 1 (Tulp1) on RPE cells and macrophages that were capable of enhancing their phagocytic capacities (Caberoy et al. 2010). Even more recently, a gene array analysis on mouse ageing retina showed genes linked to phagocytosis and phagocyte recruitment, tissue stress/injury and immune response/complement activation (complement factors B, C3, chemokine (C-C) motif ligands 2 and 12, calcitonin receptor, TLR 2, 4 and 6) (Chen et al. 2010).

The eye being an ‘immuno-priviledged’ organ in which microglia serve to keep immunological tolerance has been undermined lately by the fact that the eye is susceptible to immune-mediated diseases including AMD. The retina proper does not have professional DCs except in the peripheral regions (Forrester et al. 2010). However, CD11b+CD11c+ DCs and macrophages do lie in close apposition to the basal surface of RPE cells, and the DCs are capable of inserting dendritic processes towards the RPE layer for ‘sampling’ (auto)antigens (Forrester et al. 1994). This can trigger activation of inflammatory macrophages and induction of focal abberant angiogenesis, especially if it is located near the fovea (Xu et al. 2007).

Macrophages and DCs use different surface receptors for clearing apoptotic cells – the former involving the Axl/Mertk/Tyro3 receptor tyrosine kinase family and the latter relying on Axl and Tyro3 only (Seibenhener et al. 2007). These are different from the nonprofessional RPE phagocytes, requiring Mertk for ingestion of apoptotic material. With the recent advances in studying and precisely describing different cell death modalities, the interaction of professional and nonprofessional phagocytes with these dying cells in the retina could make AMD even more complex than we initially thought.

Taken together, chronic oxidative stress and inflammation and increased accumulation of waste materials inside and outside of RPE cells may trigger VEGF-derived choroidal neovascularisation process in certain cases.

RPE-derived VEGF in the neovascularization process

The most potent member of the VEGF family, VEGF (also called VEGF-A), was discovered in highly vascularized tumours in 1983 (Senger et al. 1983). After that, four other members in the human VEGF family have been identified: VEGF-B, VEGF-C, VEGF-D and placental growth factor (PlGF) (Maglione et al. 1991; Olofsson et al. 1996; Joukov et al. 1997; Achen et al. 1998). Vascular endothelial growth factor is a 46-kDa homodimeric glycoprotein with several isoforms including VEGF121, VEGF145, VEGF165, VEGF189 and VEGF206 generated by alternative mRNA splicing from the same gene (Ferrara et al. 1991). Vascular endothelial growth factors synthesis is strongly upregulated by hypoxia, and binding of VEGF to VEGF receptors has been shown to promote endothelial cell migration and proliferation, playing a crucial role in both normal and pathological angiogenesis. In addition, VEGF increases vascular permeability, which may also contribute to angiogenesis and tumour growth (Senger et al. 1990). Furthermore, it has a significant role in embryogenesis thus heterozygous deletion of the VEGF gene results in embryo death between days 8.5 and 9.5 (Carmeliet et al. 1996).

Vascular endothelial growth factor exerts its biological activities through multiple receptors: VEGFR-1 (Flt-1), VEGFR-2 (Flk-1/KDR) and VEGFR-3 (Flt-4), which are expressed predominantly in endothelial cells, and to a lesser extent on monocytes and macrophages (Terman et al. 1992). The binding of VEGF to its receptors initiates a signal transduction cascade mediating vascular permeability and endothelial cell proliferation and migration. Normal human choriocapillaris expresses all VEGF receptors in the choriocapillaris endothelium next to the RPE layer, suggesting a paracrine relation between RPE cells and the choriocapillaris (Blaauwgeers et al. 1999). VEGFR-2 is the major mediator of mitogenesis of endothelial cells. Through VEGFR-1, VEGF has been shown to promote assembly of endothelial cells into tubes. However, signalling via VEGFR-1 is ligand dependent; it may enhance proangiogenic effects via indirect VEGFR-2 activation or act as a negative modulator of angiogenesis induced by VEGFR-2 signalling (Gille et al. 2001).

In adults, RPE cells appear to be the only source of VEGF in the back of the eye. In the adult mouse, RPE produces VEGF164 and VEGF120 at their basolateral side towards the choriocapillaris but not VEGF188, suggesting that different VEGF isoforms may have different functions in ocular tissues and diseases (Saint-Geniez et al. 2006). In vitro, hypoxia markedly increased VEGF secretion by RPE cells at their basolateral side (Blaauwgeers et al. 1999). Similarly, transgenic VEGF188/188 mice lacking the other isoforms of VEGF showed a progressive degeneration characterized by choriocapillaris atrophy and RPE loss in aged animals. Increased photoreceptor apoptosis in aged mice led to a decline in visual acuity as detected by electroretinogram (Saint-Geniez et al. 2008). Furthermore, VEGF165 is believed to be highly associated with the blood–retina barrier breakdown and pathologic intraocular neovascularization (Ferrara et al. 2003). Instead, VEGF120 is the main isoform expressed in mouse CNV membranes (Akiyama et al. 2005).

Vascular endothelial growth factor has also been shown to have survival function in the retina (Shima et al. 2004). Furthermore, development of the choroidal vasculature is preceded by a peak of VEGF production by the RPE (Yi et al. 1998). In addition, VEGF expressed by RPE has a role in the maintenance of the adult choriocapillaris via stimulating the formation of fenestrations (Kim et al. 1999). Experimental studies have shown that RPE cell death causes loss of fenestrations and leads to choriocapillaris atrophy (Korte et al. 1984). This has also been observed in the eyes of the patients with AMD, leading to hypoxia of the outer retina (Ramrattan et al. 1994). Hypoxia causes increased secretion of VEGF by RPE, and in the presence of localized defects in Bruch’s membrane, caused by atrophy or activity of macrophages, CNV may be initiated (Blaauwgeers et al. 1999).

The role of other VEGFs in the back of the eye remains unclear. Hypoxic human RPE cells have shown to produce PlGF (Ohno-Matsui et al. 2003). Both VEGF-C and VEGF-D have been found in subretinal vascular membranes of AMD patients. It has been shown that cell–cell adhesion and cell attachment regulate VEGF-D mRNA expression in vitro, suggesting that the breakdown of these interactions might cause the overexpression of VEGF-D in human RPE (Ikeda et al. 2006).

Conclusion

  1. Top of page
  2. Abstract.
  3. Introduction
  4. RPE in the Pathogenesis of AMD
  5. Protein Degradation Processes in RPE
  6. Conclusion
  7. References

Retinal pigment epithelium cell degeneration is one of the central hallmarks in the pathogenesis of AMD. Age-related alterations in RPE cells are contributed by accumulation of lysosomal lipofuscin and extracellular drusens. A decreased capacity to remove damaged cellular proteins in RPE cells during ageing has been strongly implicated in the development of AMD. Many RPE cell clearance units seem to work in collaboration and are regulated by specific proteins. Therefore, there are increasing challenges to find novel drugs to decrease lipofuscinogenesis and to maintain effective clearance systems in RPE cells for slowing or reversing the progress of AMD.

References

  1. Top of page
  2. Abstract.
  3. Introduction
  4. RPE in the Pathogenesis of AMD
  5. Protein Degradation Processes in RPE
  6. Conclusion
  7. References