**2. Underlying stresses in the visual unit leading to ageing and pathophysiology**

Photoreceptor physiology is dependent on the supportive roles provided by the RPE and Bruch's membrane. Inherent stresses in these compartments lead to morphological and functional deterioration manifesting as 'normal' ageing changes but in the advanced ageing scenario of AMD culminate in the transition to pathology. The stresses within each compartment of the visual unit will be identified, providing therapeutic targets for intervention.

#### **2.1 Stresses generated in photoreceptor cells**

The photoreceptor is a highly specialised neuronal cell capable of detecting a single photon of light. Absorption of light by rhodopsin (R) present in the outer segment disc membranes leads to isomerization of the 11-cis retinal chromophore to its all-trans form (AT-RL), producing activated rhodopsin (R\*). Amplification of the light signal begins by rapid lateral diffusion of R\* over the disc membrane

#### *Saponin-Mediated Rejuvenation of Bruch's Membrane: A New Strategy for Intervention… DOI: http://dx.doi.org/10.5772/intechopen.96818*

and interaction with many transducin molecules. Such high mobility of R\* requires a very fluid membrane conferred by the high level of unsaturated docosahexanoic acid (DHA) in its membrane phospholipids. Further enzymatic amplification of the light signal by the guanalate cyclase-phosphodiesterase system leads to closure of sodium channels in the outer segment membrane resulting in hyperpolarisation of the cell and concomitant modulation of transmitter release. To meet the energy demands of these processes, the photoreceptor maintains the highest rates of oxidative metabolism of any cell in the body. Associated with this activity is the release of damaging oxygen radicals by the mitochondrial electron transport chain.

The presence of toxic retinoids, highly unsaturated fatty acids, high oxygen tension, high oxidative metabolism, and light is an explosive mixture for the generation of free-radical mediated damage. Since released AT-RL is highly toxic, it is rapidly reduced to all-trans retinol. However, AT-RL can react with phosphatidylethanolamine to form retinylidene-phosphatidylethanolamine (NRPE) [8]. NRPE can react with a second molecule of AT-RL to form a bis-retinoid. Further modifications produce a variety of all-trans retinal dimers including A2E, the auto-fluorescent fluorophore of lipofuscin [9]. These bis-retinoids can undergo photo-oxidation to form oxo-aldehydes which then react with proteins to form advanced glycation end-products (AGEs) that are triggers of inflammatory processes [10].

Peroxidation of polyunsaturated fatty acids such as DHA results in fragmentation of the molecule leading to a mixture of compounds that bind to proteins [11, 12]. Oxidation of DHA produces carboxy-ethyl-pyrrole (CEP)-protein adducts. Thus, oxidation of PUFAs results in lipid aggregates, lipid-protein complexes, protein cross-link formation and CEP-adducts. These CEP-adducts have been localised to the RPE and drusen and being strongly immunogenic, activate the immune system [13].

Some protection from oxidative damage is afforded by the impressive antioxidant machinery (vitamins C&E, macular pigments, and enzymes such as catalase, peroxidase, and superoxide dismutase) [14, 15]. However, this protection in photoreceptors is dependent on an adequate supply of anti-oxidants and essential metals for the enzymic system by the RPE and Bruch's membrane. Despite these protective mechanisms, considerable damage is sustained by photoreceptors. Fortunately for the photoreceptor, this damage is confined to the outer segment discs and transferred to the RPE.

Therapeutic intervention to combat this damage has been considered resulting in the Age-Related Eye Disease Study (AREDS) vitamin and anti-oxidant supplements and their effectiveness will be discussed later.

#### **2.2 Oxidative damage in the RPE**

The RPE operates in the same oxidative environment as the photoreceptor cell and therefore, the toxic reactions initiated in the outer segments will continue in the phagolysosome. Lysosomal enzymes hydrolyse the normal, undamaged protein and lipid components. recycling the base metabolites back to the photoreceptor cell. Damaged proteins, lipid-derived adducts, protein cross-links due to lipid-carbonyl attack, and aggregated lipid complexes that are no longer susceptible to lysosomal enzymes remain in the phago-lysosomal sac [16]. The lysosomal hydrolysis of bis-retinoids results in the formation of the primary age pigment, A2E. A2E and other bis-retinoids undergo further oxidation to produce a variety of toxic products that not only damage lysosomal enzymes but also damage the lysosomal membrane inhibiting the proton pumps with the subsequent increase in pH that will further diminish lysosomal enzyme activity [17].

Un-hydrolysed lipoprotein and aggregated protein complexes together with bis-retinoids are packaged and stored as the auto-fluorescent pigment lipofuscin in membrane enclosed sacs. Lipofuscin content of the RPE increases with age and can amount to nearly 20% of cytoplasmic volume in the elderly [18]. Increased oxidative stress is inferred from the accumulation of AGEs in both ageing RPE and Bruch's membrane [19]. The RPE has a battery of anti-oxidants and a robust enzymic machinery to neutralise the oxidative stress and again, the components of the protective machinery are supplied by transport across Bruch's membrane. However, the age-related accumulation of bis-retinoids and damaged proteins suggests that the anti-oxidant system is not effective in tackling this threat.

The primary functions of the RPE are (a) phagocytosis of shed outer segment discs and their degradation, (b) vectorial transport of nutrients, lipids, metals, vitamins and anti-oxidants, and the removal of waste products generated in the photoreceptor cell, and (c) fluid transport from the sub-retinal space to the choroid. The effect of the age on the various functional parameters of the RPE are poorly understood. One report has suggested that phagocytic activity is halved between the ages of 30 and 80 years [20]. Another important function of the RPE is the delivery of nutrients, anti-oxidants, vitamins, etc supplied by the choroidal circulation to the photoreceptor cell. Since the RPE is the site of the outer blood-retinal barrier, all metabolites must cross the interior of the cell to gain access to photoreceptors. Therefore, transport across the RPE is mediated by passive diffusion or facilitated by active and passive carriers in the membrane. Most active carriers utilise the sodium electro-chemical gradient generated primarily by mitochondrial respiration [21]. However, A2E generated in the RPE binds to cytochrome C of the electron transport chain impairing mitochondrial respiration and this is expected to impact on the effectiveness of active carrier transport [22, 23].

There is little information of the effect of age on the activity of ligand carriers of the RPE due largely to interference from the adjacent Bruch's membrane. This is best illustrated with the transport of retinol (vitamin A). In elderly subjects and patients with early AMD, the recovery in dark-adaptation following a strong bleach is delayed [24, 25]. This delay is thought to be due to low levels of retinoids in the RPE and therefore slower transfer of 11-cis retinal to photoreceptors for regeneration of rhodopsin. Lowered levels of retinoids in the RPE could be due to lowered uptake by the RPE itself or diminished transport of retinol across Bruch's membrane. The fact that there is improvement in dark-adaptation following vitamin A supplementation would suggest inefficient delivery across Bruch's, rather than reduced uptake by the RPE as the contributary factor [26].

Fluid transport is another important function carried out by the RPE. Retinal fluids (originating from retinal capillary beds and retinal metabolism) are transported out by the RPE predominantly by an active process [27, 28]. The daily output of fluid from the RPE has been determined to be about 0.13 ± 0.11 μl/hour/mm2 and metabolic insufficiency in the RPE would lead to fluids accumulating on top of the RPE resulting in macular oedema and/or retinal detachment [29, 30].

Therapeutic intervention in support of the RPE would require effective delivery of anti-oxidants and strengthening of its metabolic capability so as to reduce the generation of toxic products and assist in their rapid removal.

#### **2.3 Compositional changes in ageing Bruch's membrane**

Bruch's membrane mediates the exchange of nutrients and waste products between the choroidal blood supply and the RPE. An age-related compromise in these functions will reduce the capacity to supply essential nutrients to the RPE and photoreceptor cells increasing the risk of damage in these compartments.

#### *Saponin-Mediated Rejuvenation of Bruch's Membrane: A New Strategy for Intervention… DOI: http://dx.doi.org/10.5772/intechopen.96818*

The most obvious morphological change in Bruch's with age is increased thickness from about 1.5 μm in the young to 5.5 μm in the elderly [31]. This is due primarily to the deposition of normal and abnormal extracellular matrix (ECM) material. In the elderly, cross-linked and denatured (damaged) collagen accounts for nearly 50% of total collagen in Bruch's membrane [32]. There is also an increase in oxidative and non-enzymic glycosylation of proteins and lipids leading to the accumulation of toxic advanced glycation end-products, AGEs [33]. The membrane also shows an exponential increase in the level of lipid-rich debris [34]. Most of this debris arises from inefficient phagocytic processing of damaged outer segment discs in the RPE that is then extruded onto Bruch's membrane. This material then undergoes further oxidative modification with both the inherent matrix proteins and with passer-by constituents leading to further damage and deposition. Finally, the lipid components undergo free-energy driven aggregation leading to the accumulation of 100 nm diameter lipid-rich particles observed in the inner collagenous layer of Bruch's membrane [35].

Thus, in addition to the toxic metabolites mentioned above, deposits in Bruch's contain phospholipids, triglycerides, cholesterol, cholesterol esters, peroxidised lipids and apolipoproteins, immunoglobulins, amyloid, complement, and proteins specific to RPE function [36]. Heavy metal deposition has also been demonstrated that stabilises the debris in Bruch's [37].

The above changes result in gross morphological alteration of ageing Bruch's membrane that are expected to be detrimental to its transport functions (**Figure 1**).

Mechanisms exist to counteract the deleterious changes described above for Bruch's membrane. This involves the continuous synthesis and degradation of the extracellular matrix, the latter process being mediated by the matrix metalloproteinase (MMP) system [38]. Although this system performs well in the young, it deteriorates rapidly with age and more so in AMD [39].

#### **2.4 Functional deterioration of ageing Bruch's membrane**

Since Bruch's membrane is crucial for the exchange of nutrients and waste products, a deficiency in its transport functions will increase the risk of damage in the RPE and photoreceptor compartments for the reasons outlined earlier. The extent to which the compositional alterations of ageing Bruch's impact on its ability to remove

#### **Figure 1.**

*Morphology of ageing Bruch's membrane. With age, Bruch's becomes thicker and contains a lot of debris rich in lipids, and abnormal matrix and non-matrix material. The increase in thickness alone will reduce the diffusional gradients for the transport of nutrients and waste products. Vertical bar denotes the thickness of Bruch's membrane. ICL, inner collagenous layer, EL, elastin layer; OC, outer collagenous layer. Bar marker: 1* μ*m.*

fluids into the choroidal circulation, to supply adequate levels of essential nutrients, antioxidants, and vitamins to the RPE and photoreceptors, to maintain the rejuvenation potential of its membrane, and to modulate the occurrence of inflammatory responses will now be examined in both normal ageing and in the advanced ageing scenario of AMD.

#### *2.4.1 Diminished fluid transport*

The capacity for fluid transport across a membrane is designated by its hydraulic conductivity. As previously indicated, the daily output of fluid from the RPE and onto Bruch's membrane is about 0.13 ± 0.11 μl/hour/mm<sup>2</sup> . To effectively transport this amount of fluid, Bruch's needs to have a minimum hydraulic conductivity of 0.65 x10−10 m/s/Pa, and this level is referred to as the failure threshold [40, 41]. If hydraulic conductivity falls below this level, then fluid will accumulate on top of the membrane leading to a RPE detachment. Hydraulic conductivity of human Bruch's has been determined in 56 donors spanning the age range 1-91 years (**Figure 2**, modified from reference [40]). Conductivity was shown to decline exponentially with age and in the semi-log plot, the transformation is shown as a straight line. The half-life of the decay process was 16 years, i.e., conductivity was halved for every 16 years of life. Excess capacity is present in the younger population but with age, there is a drift towards the failure threshold. Extrapolating the straight line shows that the shelf-life of human Bruch's is about 123 years, but in the data of **Figure 2**, two of the normal donors have already reached the failure threshold. Bruch's from AMD donors showed a faster rate of decline in hydraulic conductivity [40] and as such, complications of RPE detachment are observed in about 12-20% of AMD patients [42].

For an effective therapeutic intervention in AMD, the exponential decay line in **Figure 2** needs to be elevated so as to avoid the failure threshold within the life-time of an individual.

**Figure 2.**

*Semi-logarithmic plot to show the exponential decay in the hydraulic conductivity of human Bruch's with age. (Modified from reference [40]).*

*Saponin-Mediated Rejuvenation of Bruch's Membrane: A New Strategy for Intervention… DOI: http://dx.doi.org/10.5772/intechopen.96818*

#### *2.4.2 Diminished metabolite and waste transport*

Metabolites ranging from the simple sugars and amino acids to the much larger lipo-protein complexes are released from the fenestrated endothelium of the choriocapillaris vessels and traverse Bruch's by passive diffusion. Most of the essential metabolites such as heavy metals, vitamins (including vitamin A), and lipids are transported bound to carrier proteins that generally have a hydrodynamic radius of about 3-12 nm.

To assess the effect of age on the diffusional status of human Bruch's membrane, a FITC-albumin test probe was utilised that has a hydrodynamic radius of about 3.5 nm, similar to most carrier proteins. Diffusional experiments were conducted in standard Using chambers utilising isolated Bruch's membrane preparations from 33 donors, age range 12-92 years. The diffusional status of Bruch's membrane was observed to decrease exponentially with age, with a half-life of 18 years (**Figure 3**) [43]. Thus, over a human life-span, diffusional status was reduced by about 10-fold. We do not know the value of the failure threshold for diffusion across Bruch's membrane. However, since most elderly subjects show delayed darkadaptation due to inefficient transport of vitamin A, the albumin diffusion values of subjects aged 77-87 years (0.024 nmol/6 mm/hour) were taken as the failure threshold.

Other in-vitro studies utilising serum proteins (MW 40-200 kDa) or FITCdextran molecules (MW 21 kDA, radius 3.3 nm) have also shown a >10-fold reduction in diffusion capability over a human life-span [44, 45].

In AMD, the reduction in diffusional transport across Bruch's membrane was much more severe compared to age-matched controls [45]. This reduction in transport is expected to impact on the nutritional and anti-oxidant support of both RPE and photoreceptor cells, increasing oxidative stress. Similarly, transport in the opposite direction i.e., removal of toxic waste products from Bruch's membrane will also be diminished leading to greater oxidative modifications and generation of further toxic products.

As with hydraulic conductivity, for effective therapeutic intervention, the diffusional decay curves should be elevated away from the failure threshold.

#### **Figure 3.**

*The effect of age on the diffusion of albumin across human Bruch's. Semi-logarithmic plot showing the decay half-life to be 18 years.*

Reduced diffusion within Bruch's membrane will also affect the protective mechanisms that depend on rapid mobility such as the interactions of complement factor H (CFH) with its many ligands and activation of pro-MMP2 in the regeneration of Bruch's. These aspects are described below.

### *2.4.3 Impaired regeneration in ageing Bruch's membrane*

The ECM of Bruch's is continuously regenerated by coupled processes of synthesis and degradation. This ensures that damaged material is removed and replaced by new ECM components synthesised by the RPE, thereby maintaining the transport integrity of Bruch's membrane. Since abnormal collagen accumulates in ageing Bruch's (amounting to 50% of total collagen in the elderly), the regeneration process appears to be dysfunctional [32]. Little is known about the synthetic rate of ECM by the ageing RPE but the accumulation of damaged ECM components suggests problems with the degradation machinery.

Matrix degradation is mediated by a family of proteolytic enzymes called the matrix metalloproteinases (MMPs). These are synthesised in the RPE and released into Bruch's membrane as latent pro-enzymes (pro-MMPs) that on activation, following the removal of a small inhibitory peptide, can degrade almost all components of the ECM [38, 46]. In Bruch's, the major MMP species are pro-MMP2 and pro-MMP9, the former being the homeostatic enzyme in the system and the latter being the inducible form. Activation of pro-MMP2 occurs on the basolateral surface of the RPE by the initial formation of a binary complex between the membrane bound MMP-14 and the tissue inhibitor of MMPs, TIMP2. This then binds pro-MMP2 to form a tertiary complex that then results in the hydrolysis of the inhibitory peptide on pro-MMP2 by a second molecule of MMP-14, to release activated MMP-2 [47].

Thus, optimal pro-MMP2 activation requires adequate levels of pro-MMP2 and TIMP2 and good mobility of these two components within the matrix of Bruch's to interact with the MMP-14 enzyme on the RPE basal membrane. The age-related reduction in diffusion within Bruch's is expected to compromise this activation potential (**Figure 3**). Furthermore, pro-MMP2 covalently binds pro-MMP9 to form the high molecular weight complex termed HMW2, reducing its level for the activation process [48]. A polymorphism in the microsatellite region of the MMP9 gene (present in most AMD patients) results in elevated levels of pro-MMP9 in both plasma and Bruch's membrane, increasing the potential for further sequestration of pro-MMP2 from the activation step [39, 49–51].

The gross alterations of ageing Bruch's membrane together with the reduction in diffusional competence are expected to hamper the mobility of pro-MMP2 and TIMP2, diminishing the activation of this MMP. Thus, levels of activated MMP2 decrease with age, and in AMD, the level was reduced by 50% compared to agematched controls [39]. It should also be noted that activated MMP2 may not be able to diffuse adequately to interact with its substrate and in the gross morphology of **Figure 1**, may be trapped within the membrane. The decreased turnover of Bruch's leads to the deleterious morphological and functional changes described earlier culminating in diminished support of RPE and photoreceptors.

#### *2.4.4 Ageing and increased susceptibility to inflammatory intervention*

Many of the toxic products produced in the RPE and present in Bruch's (as outlined earlier), including A2E, bis-retinoids, malonaldehyde, carbonyl lipids, C-reactive protein, etc., are capable of activating the complement system [52]. Thus, in elderly subjects, the exponential increase in A2E in Bruch's may be associated with a low-grade complement activation [53, 54]. A low-grade inflammatory

*Saponin-Mediated Rejuvenation of Bruch's Membrane: A New Strategy for Intervention… DOI: http://dx.doi.org/10.5772/intechopen.96818*

response may be beneficial for eliminating toxic metabolites present in Bruch's or in drusen and may serve to prevent the transition from normal ageing to pathology. Thus, the presence of the membrane attack complex and other complement factors in drusen and inter-capillary columns may allow their removal by macrophages [55].

However, indiscriminate activation of the complement system can lead to a chronic inflammatory response damaging RPE and photoreceptor cells. CFH (a 155 kDa glycoprotein) plays important roles in modulating the activation of the complement cascade. Firstly, it can bind to the toxic entities to prevent complement activation and secondly, by binding to the C3b complement component, block the progression of the cascade [56–58].

Levels of CFH in Bruch's are maintained by synthesis in the RPE and binding to glycosaminoglycans in the membrane, and delivery of plasma-derived CFH. With age, and under oxidative stress, the production of CFH by the RPE is reduced [59, 60]. Similarly, the nearly 10-fold decrease in diffusion across elderly Bruch's is expected to compromise delivery from the blood. Furthermore, in the presence of inflammatory activity in Bruch's, CFH is nitrated [61]. This nitrated CFH does not bind lipid peroxidation products nor C3b, diminishing its protective ability. Also, plasma levels of nitrated CFH are elevated in AMD patients and this may contribute to AMD progression [61].

A polymorphism in the CFH gene (Tyr402His) has been detected in about 50% of AMD patients [62, 63]. This mutated CFH shows diminished binding to toxic ligands such as malondialdehyde and C-reactive protein, and thus becomes ineffective in modulating the inflammatory response [64–66]. Mutated CFH also shows poor binding to heparin sulphate in Bruch's and hence its enhanced presence in Bruch's is compromised [67].

Ageing changes in Bruch's and the RPE therefore compromise the protective effects of CFH and in the aged AMD patient may exacerbate the inflammatory response leading to the death of RPE and photoreceptors.

## **3. Requirements for effective therapeutic intervention in dry AMD**

Oxidative damage in the RPE and Bruch's membrane is the primary driver of ageing changes in the normal elderly and more so in patients with AMD. These ageing changes diminish the supply of key anti-oxidants and vitamins required to combat oxidative stress and therefore a vicious cycle is set-up that leads to the degenerative changes in AMD.

Anti-oxidant and vitamin supplement regimes have been devised as a possible interventionist measure to reduce oxidative stress and hopefully slow the progression of the disease. Thus, the AREDS dietary supplementation cocktail was devised (vitamin C (500 mg), vitamin E (400 IU), beta-carotene (15 mg), zinc oxide (80 mg), and cupric oxide (2 mg)) and initial results showed it to be effective in reducing the risk of visual loss [68, 69]. The supplement was further modified (as the AREDS 2 formulation) by removing beta-carotene and adding lutein (10 mg) and zeaxanthine (2 mg) but did not confer any additional benefits [70].

Despite the wide use of AREDS supplements for over 10 years, controversy remains as to its usefulness since it does not prevent legal blindness in advanced AMD [71]. It has been pointed out that the earlier reported decrease in progression was related to the occurrence of neovascularisation rather than slowing the progression of dry AMD [72].

Given the fact that the diffusion of metabolites across Bruch's membrane is reduced by nearly 10-fold in the elderly, and perhaps more so in AMD, one must question the likely effectiveness of such dietary supplementation. The major problem with supplementation therapies is that they do not address transport in the opposite direction across Bruch's membrane i.e., the removal of toxic metabolites that are the likely triggers of neovascular and inflammatory episodes.

For effective therapeutic intervention, it would be ideal to improve the bi-directional transport pathways across Bruch's membrane, to improve nutritional and anti-oxidant delivery and to remove toxic waste products. This would require the destabilisation and dispersal of the lipid-rich debris and the removal of normal and damaged proteinaceous deposits. Such a strategy would also release trapped activated MMP enzymes that could participate in hydrolysing the altered collagenous components. The expected improvement in intra-membrane mobility would favour greater activation of pro-MMP2, kick-starting the normal rejuvenation machinery in Bruch's membrane. Therapeutic success would be realised if the transport decay curves shown in **Figures 2** and **3** could be elevated so that they no longer crossed the failure threshold within the lifetime of an individual. The potential implementation of such a strategy using saponin molecules is discussed next.
