**3. Subretinal deposit formation and progression: Pathogenic mechanisms**

The accumulation of specific deposits under the RPE is a very prominent histopathologic feature of eyes with AMD [24-27]. Sub-RPE deposits were first described nearly 160 years ago and were generically termed "drusen" when observed by ophthalmologists upon clinical examination of the retina [28]. Histopathological examination defines three main types of sub-RPE deposits on the basis of thickness, content, and locationt: a) basal laminar deposits (BLD), b) basal linear deposits (BLinD), and c) nodular drusen [14,29,30]. BLD and BLinD are both diffuse deposits that accumulate on opposite sides of the basal lamina of the RPE (Fig. 2); therefore, the RPE basement membrane is the crucial dividing line that demarcates the location of BLD from BLinD [14]. BLD is seen as amorphous material of intermediate electron density between the plasma membrane and the basement membrane of the RPE, often containing wide-spaced collagen, patches of electron-dense fibrillar or granular material, and occasion‐ ally, membranous debris [31]. They are distributed throughout the retina, including the periphery as well as the macula, underlying not only cones but rods as well. BLinD are diffuse, amorphous accumulations within the inner collagenous zone of BrM, external to RPE basement membrane (Fig. 2), with similar content variations [14]. BLinD are characterized by coated and non-coated vesicles as well as some membranous and empty profiles [14]. Biochemically, deposits contain phospholipids, triglycerides, cholesterol, cholesterol esters, unsaturated fatty acids, peroxidized lipids, and apolipoproteins [24-26].

Nodular drusen are discrete, dome-shaped deposits within the inner collagenous zone of BrM (i.e., external to the RPE basal lamina), which are often contiguous with BLinD, and can be difficult to distinguish from BLinD without electron microscopy [5]. Nodular drusen may also specifically contain vitronectin, immunoglobulins, amyloid, complement, and proteins associated with RPE cell function [32] as well as other poorly characterized components [24-27]. Further, low-grade monocyte infiltration within the choriocapillaris is often present underlying areas of deposits [25,27,33].

is the elastic fiber layer (Fig. 3) sandwiched between the inner and outer collagenous layers. Finally, two basal laminas define the innermost and outermost layers. The innermost layer is the basal lamina of the RPE; the outermost layer is formed by the basement membrane of the endothelial cells that comprise the choriocapillaris [6,18,21]. The collagen layers contain striated collagen fibers mainly type I, III, and V [6,18]. Type I confers tensile strength to the tissue, type III is normally present in tissues with elastic properties, and type V acts to anchor basement membranes to stromal matrixes [17,18]. The interfiber matrix of BrM is comprised largely of glycosaminoglycans such as heparan sulfate (25%) and chondroitin/dermatan sulfate (75%). These provide an electrolytic barrier to diffusion and serve important regulatory roles by binding extracellular proteins and growth factors that are vital for cellular processes such

42 Age-Related Macular Degeneration - Etiology, Diagnosis and Management - A Glance at the Future

The basement membrane of both the choriocapillaris and RPE are comprised of mostly type IV collagen [17,18]. The basement membranes additionally contain laminin, heparan sulfate, proteoglycans, and fibronectin. The outermost basal lamina also incorporates type VI collagen, which is associated with the choriocapillaris [17,18]. It has been suggested that this collagen

**3. Subretinal deposit formation and progression: Pathogenic mechanisms**

The accumulation of specific deposits under the RPE is a very prominent histopathologic feature of eyes with AMD [24-27]. Sub-RPE deposits were first described nearly 160 years ago and were generically termed "drusen" when observed by ophthalmologists upon clinical examination of the retina [28]. Histopathological examination defines three main types of sub-RPE deposits on the basis of thickness, content, and locationt: a) basal laminar deposits (BLD), b) basal linear deposits (BLinD), and c) nodular drusen [14,29,30]. BLD and BLinD are both diffuse deposits that accumulate on opposite sides of the basal lamina of the RPE (Fig. 2); therefore, the RPE basement membrane is the crucial dividing line that demarcates the location of BLD from BLinD [14]. BLD is seen as amorphous material of intermediate electron density between the plasma membrane and the basement membrane of the RPE, often containing wide-spaced collagen, patches of electron-dense fibrillar or granular material, and occasion‐ ally, membranous debris [31]. They are distributed throughout the retina, including the periphery as well as the macula, underlying not only cones but rods as well. BLinD are diffuse, amorphous accumulations within the inner collagenous zone of BrM, external to RPE basement membrane (Fig. 2), with similar content variations [14]. BLinD are characterized by coated and non-coated vesicles as well as some membranous and empty profiles [14]. Biochemically, deposits contain phospholipids, triglycerides, cholesterol, cholesterol esters, unsaturated fatty

Nodular drusen are discrete, dome-shaped deposits within the inner collagenous zone of BrM (i.e., external to the RPE basal lamina), which are often contiguous with BLinD, and can be difficult to distinguish from BLinD without electron microscopy [5]. Nodular drusen may also specifically contain vitronectin, immunoglobulins, amyloid, complement, and proteins

as adhesion, migration, and differentiation [17,22,23].

acids, peroxidized lipids, and apolipoproteins [24-26].

may act to anchor fenestrated capillaries to the underlying choroid.

Clinically, deposits of AMD are classified on fundoscopic features of morphology and size [24]. Although multiple classifications exist, most clinicians use size to classify drusen: small or "hard" (<63 μm) and soft, intermediate (>63 to <125 μm) and large (>125 μm) drusen [24]. When hard or soft drusen coalesce to the point of losing their boundaries they are then classified as "diffuse." Although diagnosis of AMD is typically made when intermediate or large drusen are present, the diagnosis can be also be made in the absence of drusen based on the presence of pigmentary changes indicative of RPE degeneration [3,29,30]. The specific contribution of drusen to AMD complications and progression are not well characterized, but the presence of macular drusen is considered a strong risk factor for the development of both forms of late AMD, geographic atrophy and neovascular AMD [3,34,35]. In general, eyes with clinical AMD have been found to express all three deposit subtypes [14,25,36]. Furthermore, histological, immunohistochemical, and ultrastructural studies of surgically-excised choroidal neovascular (CNV) membranes have shown that the cellular and extracellular constituents of CNV are the same regardless of the underlying disease, with the exception of the amount of BLD and the presence of BLinD, which is virtually exclusively found in CNV specimens from patients with AMD [29,37].

The cellular and molecular events involved in drusen formation have not been fully elucidated. Lack of scientific consensus exists regarding the origin of drusen, but at least five different paradigms are currently proposed to explain deposit formation in AMD; a) genetic hypothesis, b) lysosomal failure/lipofuscin hypothesis, c) choroidal hypoperfusion hypothesis, d) barrier hypothesis, and e) RPE injury hypothesis. Because our research is based on the RPE injury hypothesis, in this section this theory is reviewed in light of the more recent finding.

There are a number of direct and/or indirect lines of evidence supporting a role for the RPE in drusen biogenesis. According to traditional models of drusen formation, any cellular material residing within drusen is predicted to be of RPE origin. Indeed, RPE-derived basal laminae, organelles and cellular fragments, and even entire cells can be detected in early "drusen". Some authors have described the appearance of RPE "debris" blebbing into drusen or pre-drusen sites [38]. RPE constituents, such as basal laminae, as well as lipofuscin and melanin granules, are observed within early drusen, where they likely contribute to drusen volume and formation.

The theory that drusen were derived from damaged RPE was originally postulated by Donders, who first described drusen in a post-mortem eye, believed that drusen were derived from RPE nuclei, based on the supposition that the latter are relatively resistant to degradation [39]. Donders' theory was later modified by De Vicentis (1887) who proposed that degenerative change in the RPE cytoplasm, rather than in the nucleus, was the precipitating event. On the other hand, Muller (1856) proposed that drusen result from aberrant secretion of basement membrane components by the aged RPE [40].With the advent of electron microscopy, the substructural features of drusen were revealed, and new variants of the earlier theories were advanced. Some investigators have concluded, that drusen are formed when the RPE expels portions of its basal cytoplasm into BrM [38], possibly as a mechanism for removing damaged cytosol [41] or as a byproduct of phagocytic degradation [19]. Others have postulated that drusen are formed by autolysis of the RPE, due to aberrant lysosomal enzyme activity [42], although enzyme histochemical studies failed to demonstrate the presence of lysosomal enzymes in drusen [43]. Additional mechanisms for drusen formation, including lipoidal degeneration of the RPE, have been proposed [44,45].

recruitment may be beneficial or harmful depending upon their activation status at the time of recruitment [50,61]. Nonactivated or scavenging macrophages may remove deposits without further injury. Activated or reparative macrophages, through the release of inflam‐ matory mediators, growth factors, or other substances, may promote complications and progression to the late forms of the disease [37,50,62,37]. As discussed below, the effect of

Cigarette Smoking and Hypertension Two Risk Factors for Age-Related Macular Degeneration

http://dx.doi.org/10.5772/53958

45

**Figure 4.** Schematic drawing of the early stages of age-related macular degeneration (AMD). First stage: Oxidant in‐ jury at the retinal pigment epithelium (RPE) causes bleb formation and decreased matrix metalloproteinases (MMPs). Blebs than accumulate between the RPE and the basement membrane as basal laminar deposits (BLDs). Second stage: Increased matrix turnover characterized by increased MMPs and decreased collagens. BLDs progress into basal linear deposits (BLinDs) and drusen and are located within the inner collagenous layer of Bruchs membrane. Third stage: Macrophages recruited to the site of injury by chemotactic factors are responsible for release of cytokines, angiogenic factors, and oxidants that perpetuate RPE injury and lead to late stages of AMD. Abbreviations: MPO = myeloperoxi‐ dase; NF-κB = nuclear factor kappa-light-chain-enhancer of activated B cells; TNF-α: tumor necrosis factor-alpha;

It has been postulated that environmental oxidants are frequently implicated in RPE injury and may contribute to deposit formation. Cigarette smoking is the strongest environmental risk factor for all forms of AMD, even in people exposed to passive smoking [63-66]. People who have smoked at least 100 cigarettes (lifetime) have approximately triple risk of developing AMD compared to individuals who have never smoked. Current smokers and heavy smokers

MCP-1: monocyte chemotactic protein-1; VEGF: vascular endothelial growth factor.

**5. Smoking and age-related macular degeneration**

have even higher AMD risk.

cigarette smoking and Ang II in the mentioned stages will be reviewed in depth.

In the modern version, the RPE injury hypothesis proposes that deposit formation is secondary to chronic, repetitive but nonlethal RPE injury [46, 47]. Two separate phenomena must be distinguished: the injury stimulus and the cellular response. The most widely implicated injury stimuli are various oxidants, especially those induced by RPE exposure to visible light or those derived from endogenous metabolism [48,49]. More recently, inflammatory-derived injury stimuli have also become implicated, including oxidants, complement, immune complexes and factors produced by macrophages or monocyte [50-52]. Inflammatory cells might be responsible for drusen progression into CNV by secretion of growth factors and cytokines that will damage the choriocapillaris and stimulate the invasion of neovessels into the subretinal space [37,51,52].

Irrespective of the injury, this model proposes that all stimuli result in a final common pathway of cellular responses that cause the actual deposits. Cellular responses that can lead to deposit formation include RPE cell membrane blebbing and dysregulation of extracellular matrix (ECM) production and breakdown. Accumulation of sub-RPE blebbed material in the setting of imbalanced breakdown and resynthesis of basement membrane and BrM ECM is proposed to produce the various deposits of AMD. Repetitive injury ultimately can kill RPE, leading to late dry AMD [6].
