In vitro models

Although RPE cells have been successfully grown for many years, it is only recently that in vitro systems have been used to model AMD.

Importantly, primary cultures were successfully maintained from CNV membranes from AMD eyes (Schlunck et al., 2002), this will enable new approaches to study genetical aspects of the role o f the RPE in AMD. More recently and published in abstract form only is a study replicating the RPE-Bmch’s membrane-choriocapillaris complex. This utilises human amniotic membranes represnting Bruch’s membrane, ARPE 19 cells and human umbilical vein endothelial cells representing the

choriocapillaris (Hamilton et al., 2003). This lack of adequate in vitro model led to the development o f part o f this project.

The use of in vitro cultured cells over in vivo human or animal eyes has several advantages:

1. Human tissue is in short supply (moral and ethical issues), and cell culture can be used to amplify the number of cells needed for the application of molecular and biochemical techniques.

2. Cultured cells can be expanded and maintained in culture for long periods of time.

3. Cell culture provides strict control of the environment and greater experimental flexibility and allov^s easy access to the basolateral membrane.

4. There is a potential for cultured RPE to be used in transplantation strategies for the treatment of retinal disease, and understanding the physiology o f cultured RPE may prove vital to the success o f this work.

However in vitro models have particular disadvantages, which include:

1. Phenotypic variability (Burke et al., 1996); Cultured RPE display morphological differences from those in vivo.

2. Loss o f polarised expression of the Na"^/K^ pump and the expression o f ion channels that differ from those present in native RPE (Rizzolo, 1990; Nabi et al.,

1993)

3. Culture conditions and donor age passage number may be critical factors that affect the cell biology of RPE cells in culture.

An ideal in vitro model of sub-RPE deposits similar to those seen in AMD would consist of a well-characterised and stable RPE cell line expressing a differentiated phenotype that resembles closely that found in vivo.

In general, anchorage dependant cell culture requires at least two components of interaction with the substrate for adequate differentiation, especially when an in vitro

model is being created. Firstly, adhesion is required to allow the attachment and spreading necessary for cell proliferation and secondly specific interactions involving the basement membrane and ECM are required. Differentiation can be morphological, such as tight junction or apical microvilli formation, or functional, such as migration or phagocytosis.

1.7 ECM and regulation of its composition

The ECM is a complex meshwork of proteins and carbohydrates, which is synthesised and deposited in the vicinity o f the cells, forming an organised intercellular network. The matrix not only provides a mechanical support where cells reside, but can interact directly with cells, controlling cell shape, proliferation, migration and differentiation (Alberts et al., 1994). Many of the molecular events that define the interaction between cells and the ECM are still largely unknown. However an increase in our knowledge of this interaction has come from the elucidation of the primary sequence o f several ECM proteins and the precise location and characterisation o f their binding domains. The two main classes o f macromolecule in the ECM are glycosaminoglycans (GAG) and fibrous proteins. GAGs occupy a large amount o f space and form hydrated gels. They are termed proteoglycans when linked to a protein. Fibrous proteins are o f either structural type such as collagen and laminin or adhesive type such as fibronectin.

In the eye, the RPE and its basement membrane rest upon a highly organised ECM, Bruch’s membrane (Marmor and Wolfensberger, 1998)(chapter 34). This structure is visible by light microscopy and its increased thickness has been associated with both pathology and ageing as described in section 1.2.2/1.3.

The regulation of ECM is critical to a variety of important biological processes involving signalling pathways. Degradation is controlled by the metalloproteases (MMPs) and the serine proteases, and their inhibitors, the tissue inhibitors o f the metalloproteases (TEMPs) and the serine protease inhibitors or serpins (Alberts et al.,

1994).

1.7.1 The role of matrix metalloproteinases and their inhibitors in AMD and ageing

There is increasing evidence that tissue inhibitors of metalloproteinases and matrix metalloproteinases themselves have a role to play in the health and disease of the retinal pigment epithelium. MMPs are structurally related zinc metalloproteinases. To date at least eighteen human MMPs have been identified. They are either secreted from cells or bound to the cell surface (Massova et al., 1998; Sethi et al., 2000). Secreted MMPs include collagenases, gelatinases, stromelysins and matrilysins, metalloelastase, enamelysin and epilysin. There are six membrane-type MMPs (MT-MMPs) that are considered to participate in pericellular proteolysis.

are inducible. Their effectors include growth factors, cytokines, physical stress such as heat shock, UV irradiation and mechanical stress, oncogenic transformation, and cell­ cell and cell-matrix interactions (Nagase and Woessner, 1999).

The family o f TIMPs includes four distinct gene products that are relatively specific inhibitors of the MMPs, all with molecular weights in the range 22-29 kDa. The local balance between MMPs and TIMPs plays a major role in extracellular matrix remodeling during development and in disease at many sites. Unlike the other TIMPs, which are soluble, TIMP 3 is unique in being an insoluble component o f the extracellular matrix. MMP 1, 2, 3 and 9, and TIMP 3 transcripts have been demonstrated in cultured human RPE, choroidal microcapillary endothelium and pericytes (Vranka et al., 1997). Chong et al have identified the presence of TIMP 3 in Bruch's membrane and the elastic layer of large choroidal vessels (Chong et al., 2000). Immunohistochemistry experiments by Fariss and colleagues have found TIMP 3 in Bruch’s membrane (Fariss et al., 1997), whereas Guo et al have identified MMP 1, 2, 3 and 9 in Bruch's membrane (Guo et al., 1999) by immunohistochemistry and Western blot analysis.

The following observations support, or are consistent with, the hypothesis that modulation o f the MMP/TIMP balance is important in ageing and the pathogenesis of AMD.

1. Immunohistochemistry, Western blot analysis and reverse zymography have shown an increase in TIMP 3 protein in Bruch’s membrane with AMD and age (Kamei and Hollyfield, 1999).

2. There is an increase in MMP 2 and MMP 9 proteins in Bruch’s membrane/ choroid with age (Guo et al., 1999).

3. MMP 2 is increased in the ECM of photoreceptors in AMD (Plantner et al., 1998).

4. Sorsby’s fundus dystrophy is a disease caused by mutations in the TIMP 3 gene that resembles AMD. In particular this disease causes patients to produce BLamD in large quantities (Weber et al., 1994a).

5. Abnormalities of the ECM may promote a pro-angiogenic RPE phenotype that contributes to the development of angiogenesis (Campochiaro et al., 1999), an important aspect of AMD.

Further details of the role of metalloproteinases in the eye can be read in the review by Sethi et al (Sethi et al., 2000).

1 ,7.1,1 The MMP/Cytokîne Connection

In addition to modifying extracellular matrix proteins, MMPs are intimately involved in the regulation of the activities of cytokines and cytokine receptors. The MMP axis has several areas of overlap with the cytokine network (Sivak and Fini, 2002).

Inflammatory cytokines or growth factors can regulate the expression of MMPs (Eichler et al., 2002). Cytokine activation of cells can also lead to increased processing of MMPs from the inactive form to the active enzymes (Eichler et al., 2002). Cytokines and their receptors can also be substrates for MMP action, for example; the pro-inflammatory c>tokine IL-1 can be cleaved and inactivated by MMP 1, 2, 3, and 9 (Ito et al., 1996). At this point it is interesting to note that sub-RPE deposits, specifically drusen, have been shown to regress following laser treatment (Choroidal Neovascularization Prevention Trial Research Group, 1998; Friberg, 1999; Folk and Russell, 1999). Drusen regression occurs even remote to the site o f the laser bums. The mechanism o f laser- induced drusen regression is not known but one possibility is that it provokes a low- grade inflammatory response. As laser treatment has been shown to cause increased levels o f TNF-a production (Morimura et al., 2001; Bradley et al., 2000) a reduction in drusen may be associated with increase TNF-a. In addition TNF-a, is a pro- inflammatory cytokine and has been reported to activate MMP-2 (Han et al., 2001) (Gearing et al., 1994). These observations led the experiments described in section 5.7.