**11. Lipoproteins, apolipoproteins and the retina**

368 Lipoproteins – Role in Health and Diseases

strongest association with AMD.

lymphocytes; transport of lipids within the brain.[53]

phases of the reinnervation process.[83;84]

lipid-lipoprotein levels.[87]

metabolising enzymes, including lipoprotein lipase, and LCAT.

**10. Apolipoprotein E** 

controls.[78] The evidence to date suggests that, of the apolipoproteins, ApoE has the

ApoE is a structural component of plasma chylomicrons, VLDL, and a subclass of HDL. It is a 299 amino-acid protein, and is synthesised in a large number of tissues including the spleen, kidneys, lungs, adrenal glands, liver, brain and retinal Müller cells.[81] ApoE is polymorphic, with three common isoforms: E2, E3 and E4, which are coded for by three separate alleles: Apo ε2, Apo ε3 and Apo ε4. These alleles are differentiated on the basis of cysteine-arginine residue interchanges at sites 112 and 158 in the amino acid sequence.[82] As a result of this polymorphism, six common phenotypes exist: three homozygous phenotypes (ε3ε3, ε2ε2, ε4ε4) and three heterozygous phenotypes (ε2ε3, ε2ε4, ε3ε4). ApoE is crucial to many processes, including: cholesterol transport and metabolism; receptormediated uptake of specific lipoproteins; heparin binding; formation of cholesteryl-esterrich particles; lipolytic processing of type IIIβ-VLDL; inhibition of mitogenic stimulation of

ApoE is an important regulator of cholesterol metabolism because of its affinity for ApoEspecific receptors in the liver, and its affinity for LDL receptors in the liver and other peripheral tissues requiring cholesterol.[53] ApoE-specific receptors are present on the membranes of hepatic parenchymal cells, and have a high binding affinity for chylomicron remnants, IDL and a sub-class of HDL. ApoE also regulates the activity of several lipid-

ApoE is found in greatest concentrations in the liver. However, it is also the predominant apolipoprotein in the brain, and is responsible for lipid transport and cholesterol regulation within the central nervous system (CNS). ApoE is a major component of plasma and cerebrospinal fluid, and plays a fundamental role after CNS injury, where it appears to regulate the transport of cholesterol and phospholipids during the early and intermediate

ApoE polymorphisms result in differences in the metabolism of ApoE-containing lipoprotein particles.[85] For example, it is possible that certain ApoE polymorphisms affect their ability to interact with lipoprotein lipase in the conversion of VLDL to LDL.[86] Indeed, ApoE polymorphism influences plasma lipid levels both in sedentary states and in their response to exercise, and it is therefore believed to be related to risk for coronary artery disease. In general, carriers of the Apo ε4 allele have higher levels of total cholesterol and LDL-cholesterol than those with the Apo ε3 allele. ApoE polymorphism also appears to play a role in the responsiveness of blood lipids to dietary and lipid-lowering drug interventions. Thus, the ApoE gene-environmental interactions contribute to population variance in blood

ApoE receptors also play an important role in lipoprotein metabolism. The primary physiological role of ApoE is to facilitate the binding of lipoproteins to LDL receptors, As noted previously, the ageing retina features changes in the RPE and BrM, which include changes in the lipoprotein and apolipoprotein composition of both structures. These changes may progress to the disease state of AMD. In recent times, evidence accrued from light microscopy, ultrastructural studies, lipid histochemistry, isolated lipoprotein assays, and gene expression analysis had led to the identification of many of the constituents that deposit in the RPE and BrM with age and AMD.[93] One of the universal changes that occurs with age is the development of BlamDs between the RPE and BrM.[11;12] This process may progress to the development of a 'lipid wall', mainly composed of neutral lipid deposits, decreasing the permeability of BrM and hindering metabolic activity between the RPE and BrM, preceding pathological changes associated with AMD.[10;93;94] When these deposits accumulate within the inner collagenous layer of BrM, they are referred to as basal linear deposits (BlinDs) and are a histopathological hallmark of AMD, which, when sufficiently large, can be recognised clinically as drusen.[13-15;95]

Much of the debris that accumulates in BrM in the form of BlinDs is composed of lipoproteins and lipoprotein particles.[14] It has been found that almost 60% of the total cholesterol within these lipoproteins is esterified cholesterol.[96] Furthermore, the esterified cholesterol within BrM was enriched between 16 and 40-fold compared to plasma. If these extracellular lipid deposits had been derived from plasma, more than 90% of the phospholipid would be phosphatidylcholine, whereas in actual fact, these lipoproteins are comprised of less than 50% phosphatidylcholine.[96] Indeed, the composition of drusen, which are essentially large BlinDs, has been shown to include esterified and unesterified cholesterol, and multiple apolipoproteins, including apolipoproteins B, A-I, C-I, C-II, and E, appearing with frequencies ranging from 100% (ApoE) to approximately 60% (A-I).[88;91;97;98] Interestingly, ApoC-III, although abundant in plasma, is present in fewer drusen (16.6%) than ApoC-I (93.1%), which is not present in plasma in large quantities, indicating either a specific retention of plasma-derived apolipoproteins within drusen, or an intraocular source for these apolipoproteins.[93] It is now understood that the majority of lipoproteins in BrM have undergone intracellular processing within the RPE prior to secretion as neutral lipids, mainly esterified cholesterol.[99;100] The RPE origin has been definitively shown by two groups using metabolic labelling and immunoprecipitation in ratderived and human-derived RPE cell lines that were shown to secrete full-length ApoB.[101;102] This evidence is further strengthened by the finding of microsomal triglyceride transfer protein within native human RPE, indicating that the RPE is capable of secreting lipoprotein particles.[102] The pattern of lipid deposition in BrM with age, in which debris appears firstly in the elastic layer and then fills in towards the RPE, is also consistent with this lipid being primarily of RPE origin.[103]

The hydrophobic nature of the age-related thickening of BrM has been implicated in the aetiopathogenesis of AMD. In the case of Apo E, it is noteworthy that ApoE4 presents a positive charge relative to both ApoE2 and ApoE3. ApoE4 possesses arginine at residue 112 of the amino acid sequence, whereas ApoE3 possesses cysteine at this position, and in the case of ApoE2, the most frequent variant has cysteine instead of the normally occurring arginine at residue 158. Thus, ApoE3 presents a neutral charge, and ApoE2 a negative charge, relative to ApoE4.[53] Souied *et al.* suggested that this difference in charges between the ApoE isoforms may also contribute to differences in the clearance of debris through BrM.[104]

It appears that Müller cells are the most prominent biosynthetic sources of ApoE in the neural retina, and RPE cells are the most prominent sources in the RPE/choroid.[91] However, it remains unclear whether the concentration of ApoE in the cytoplasm of some RPE cells, especially those in close proximity to drusen, is the result of biosynthesis or selective accumulation. It has been shown that, in both the central and peripheral nervous systems, ApoE expression by astrocytes is up-regulated in response to neuronal injury and neuro-degenerative disease.[84;105;106] Indeed, there is evidence for ApoE up-regulation by Müller cells in degenerating human retina, where increased ApoE immuno-reactivity is found in the sub-retinal space of detached retinas[107] and in the Müller cells of retinas affected by glaucoma or AMD.[108] Furthermore, the relatively high levels of ApoE mRNA detected in the retina, especially in the eyes of older donors and in an individual with documented AMD, support the view that up-regulation by retinal glia may be responsible for the observed increase in ApoE expression.[91]
