Análisis Estadístico Variable dependiente: Porcentaje de Remoción (%) [74]

The RPE monolayer is embryologically derived from the same neural tube tissue that forms the neurosensory retina. It is necessary for the support and viability of the photoreceptors. The cells are cuboidal in cross section and hexagonal when viewed en face. Each cell is 10-14 pm in diameter in the macula but becomes flatter and broader in the periphery. The apical side of the RPE has numerous long microvilli, which reach up and envelope the outer segments for phagocytosis (Bok et al., 1993) and also participate in retinal adhesion (Hageman et al., 1995). The mid-portion contains the nucleus, Golgi apparatus, endoplasmic reticulum and other organelles. Basal vesicles are also seen in this area and thought to be important in the health o f the RPE cell (Ishibashi et al., 1986; Orzalesi et al., 1982). The basal membrane has convoluted infoldings that increase its surface area for absorption and secretion. Table 1.2 describes the many function o f the RPE.

Table 1.2 Role of the retinal pigment epithelium cell

Role Function

Optical (pigment) Light screening

Scatter reduction / light reflection

Structural Photoreceptor support

Shed photoreceptor phagocytosis Retinal adhesion

Environmental Posterior blood-retinal barrier

Ion and water transport Nutrient transport Free radical binding

Visual cycle Retinoid binding and storage

Visual pigment regeneration Cycling of other outer segment constitutents

Trophic Ocular development and regeneration

Response to injury by repair mechanisms Table adapted from (Marmor and W olfensberger, 1998) showing the various functions o f the RPE.

1.5.1.1 RPE cytoskeleton

The ability o f eukaryotic cells to adopt a variety o f shapes and to carry out coordinated and directed movements depends on a complex network of protein filaments that extend throughout the cytoplasm called the cytoskeleton.

The RPE cytoskeleton consists of

1. Membrane cytoskeleton, which contributes to membrane contour, stability, motility and cell- cell interactions through junctional complexes such as zonulae adherens (Hitt and Luna, 1994; Path and Burgess, 1994; Gilligan and Bennett,

1993).

2. Cytoplasmic cytoskeleton, which is responsible for the structural and tensile properties o f the cell as well as intracellular transport (Hitt and Luna, 1994; Path and Burgess, 1994; Gilligan and Bennett, 1993).

The cytoskeleton is composed of three fibrous elements, actin filaments, microtubules and intermediate filaments. Actin filaments are two-stranded helical polymers of the protein actin with a diameter o f 5-9 nm. The actin cytoskeleton has structural and motile roles in the RPE. Structurally, they provide anchorage for membrane proteins, maintain epithelial integrity (Kalnins et al., 1995), cell deformability and otherwise contribute to the mechanical properties of the cytoplasm (Hitt and Luna, 1994). It supports cell projections and maintains cell shape (Clarke and Spudich, 1977). Actin filaments also interact with myosin motors to produce contraction and intracellular transport of cellular constituents (Burnside et al., 1983), cell locomotion (Campochiaro and Glaser, 1986) and phagocytosis (Besharse et al., 1982). In polarised RPE cells, actin filaments are localised to the basal infoldings, the lateral membrane cytoskeleton, apical projections and dense circumferential microfilament bundles (CMB), which encircle the cell (Nguyen-Legros, 1978; Marmor and Wolfensberger, 1998).

Microtubules are hollow, long cylinders made o f the self-assembling polymer protein, tubulin with an outer diameter of 25nm. They have a critical role in cell structure, forming a scaffold within the cell (Marmor and Wolfensberger, 1998)(chapter 3). Microtubules have been shown to be involved in fast axonal transport o f membranous organelles (Vale et al., 1985), extension of tubular lysosomes in macrophages (Hollenbeck and Swanson, 1990), and pigment granule movement (Rodionov et al.,

Intermediate filaments are rope like fibres with a diameter of lOnm and made of intermediate filament proteins, which constitute a large and heterogeneous family. The expression pattern of RPE cell intermediate filaments proteins varies with species and culture conditions. Human RPE cells express cytokeratins 8 and 18 in vivo with no vimentin, but when placed in culture they express cytokeratins 7,8,18,19 and vimentin (Hunt and Davis, 1990; McKechnie et al., 1988). The pattern in vitro is also influenced by culture conditions. For example, exposure to vitreous decreases expression o f both cytokeratins and vimentin (Vinores et al., 1990).

Integrins are only briefly mentioned at this point to illustrate the importance o f these transmembrane proteins for the ECM to communicate with the cytoskeleton (Burridge et al., 1988; Sastry and Horwitz, 1993). Integrins are the main transmembrane linker proteins, whose external domains bind to the ECM while the cytoplasmic domain is linked to the actin filaments in stress fibers. In addition integrins can activate intracellular signalling cascades (Damsky and Werb, 1992; Boudreau and Jones, 1999).

1.5.2 Melanin / lipofuscin relationship

Melanin granules are abundant in the apical and mid-portion o f the RPE (Hogan et al., 1971; Feeney, 1978). Two shapes of granules are present, ellipsoid granules (1pm in diameter to 2-3pm in length) primarily located towards the apical portion of the cell and spherical granules in the mid-portion (Hogan et al., 1971). Melanin granules are involved in: free radical scavenging, free radical generation, absorption of the effects of thermal damage with additional chemical binding and photophysical properties (Marmor and Wolfensberger, 1998)(chapter 4).

With age this distribution is lost (Hogan et al., 1971) and there is a significant decline in total numbers o f granules after the age o f 40 (Feeney-Bums et al., 1984). There is also a corresponding increase in the complex granules melanolipofuscin (melanin with a cortex of lipofuscin) and melanolysosomes (melanin with a cortex of enzyme reactive material) (Feeney, 1978). These complexes exhibit a regional distribution similar to that of lipofuscin (Marshall, 1987) with the highest density in the extrafoveal macula decreasing density towards the periphery and the fovea (Feeney-Bums et al., 1984) and are thought to represent melanin in the process o f repair, modification or degradation (Feeney, 1978).

1.5.3 Grow th factors in the RPE

Growth factors are one of the major types of signals needed for cells to maintain their structure, function and survival. They are proteins that bind to cell surface receptors and influence intracellular signal transduction pathways. Their potential role in AMD, whether the source is RPE, retina or endothelial cells of the choriocapillaris, is very wide-ranging. RPE cytokine production and cytokine induced changes have been extensively reviewed by Holtkamp et al (Holtkamp et al., 2001).

The following growth factors are o f particular relevance to this project;

1. Tumour growth factor (TOP) superfamily and their receptors have been shovm to be present in the RPE; they are important in the pro-fibrotic phase of healing and scar formation.

2. Platelet derived growth factors (PDGF) are involved in the regulation o f cell growth and differentiation (Mercola et al., 1990). In particular, in the RPE, PDGF stimulates cell proliferation (Leschey et al., 1990; Campochiaro et al., 1994) and migration (Campochiaro and Glaser, 1985).

3. Vascular endothelial growth factor (VEGF) released from the RPE cell increases permeability of microvessels and is involved in angiogenesis. RPE VEGF has been studied in vitro and in vivo (Adamis et al., 1993) in particular with reference to diabetic retinopathy (Murata et al., 1996) and the induction of proliferative diabetic retinopathy.

4. Fibroblast growth factor (FGF) members are produced by RPE cells; in particular bFGF is modulated by cell density, cell adhesion, ECM components, cellular stress and cytokines (Bost and Hjelmeland, 1993; Hackett et al., 1997). Its function is in modulation of inflammation and the immune response as well as its more traditional role as a mitogen and survival factor.

5. Epidermal growth factor (EGF) causes proliferation o f the RPE (Leschey et al., 1990; Arrindell et al., 1992).