Ординатура / Офтальмология / Английские материалы / Textbook of Vitreoretinal Diseases and Surgery_Natarajan, Hussain_2008

Textbook of Vitreoretinal Diseases and Surgery

dry AMD (Cameron et al, 2007) eyes and in promoter-based assays, this finding was not replicated in another study (Kanda et al, 2007). Thus, the precise role of the LOC387715 and HTRA1 SNPs in AMD is as yet unknown, and their interactions with different factors in the complement pathway remain speculative. However, such speculations cannot be addressed unless extensive data on gene– gene interactions in the background of other nongenetic factors leading to the pathophysiology of AMD are elucidated (Swaroop et al, 2007)

APOE

In the past few years, association of variants in genes involved in lipid metabolism such as Apolipoprotein E (APOE) have increased our understanding of the underlying mechanism of AMD development. Based on common pathogenic features including lipid deposition (drusen and plaque formation), thickening of connective tissue (Bruch’s membrane and arterial inner lining) and elevated levels of CRP in serum, a common mechanism for the development of AMD and atherosclerotic cardiovascular diseases was proposed (Freidman 2000). However, it was noted that the effect of APOE alleles on AMD risk was contrastingly different than that of atherosclerosis and cardiovascular diseases (Baird et al, 2004; Zareparsi et al, 2004). Association of APOE in AMD has been reported by several groups (Klaver et al, 1998; Schmidt et al, 2002; Baird et al, 2004; Zareparsi et al, 2004) showing a reduced risk of AMD with APOE-e2 allele and higher risk with allele APOE-e4. However, APOE polymorphisms have exhibited varied geographical and ethnic variations across AMD patients worldwide (Kaur et al, 2006).

Toll like Receptor 4 (TLR4)

A recent study by Zareparsi et al, (2005) implicated the TLR4 gene (9q32-33) in AMD pathogenesis. Toll like receptors are involved in innate immunity and pathogen recognition (Cook et al, 2004), linked to regulation of cholesterol efflux and participates in phagocytosis of photoreceptor outer segments by the RPE (Kiechl et al, 2002; Castrillo et al, 2003; Kindzelskii et al, 2004; Blander et al, 2004). The D299G polymorphism in TLR4 was associated with a 2.65 folds increased risk of AMD, thereby suggesting that altered TLR4 signaling by this variant may influence phagocytic function of RPE which in turn may contribute to RPE damage. It was also shown that TLR4-D299G had an additive effect on AMD risk (OR = 4.13, p = 0.002) with allelic variants of APOE and ATP binding cassette transporter-1 (ABCA1), which are involved in cholesterol efflux. However, the effect of TLR4, APOE and ABCA1 variants on AMD susceptibility was in contrast to that seen in atherosclerosis. But TLR4 polymorphisms have not been explored extensively across different world AMD populations compared to other candidate genes.

Mechanisms of AMD Development

Several pathogenic mechanisms have been proposed to understand the complex etiologies of AMD development including RPE cell death, oxidative damage of cellular components, mitochondrial dysfunction and accumulation of toxic components such as lipofuscin and advanced end glycation products (Haddad et al, 2006). It was thus proposed that variations in genes involved in inflammation, oxidative stress and cholesterol metabolism play significant role in the pathogenesis

8 of AMD.

Genetics of Age-related Macular Degeneration

ROLE OF COMPLEMENT ACTIVATION AND INFLAMMATION IN THE PATHOGENESIS OF AMD

Inflammation has been implicated in several diseases of aging like Alzheimer’s disease, stroke and cardiovascular disease. Interestingly all these diseases share lots of similarities with AMD with respect to pathological features and common biomarkers for the disease such as increased levels of CRP, CFH and Homocysteine in plasma of patients, presence of serum amyloid P component in drusens (the levels of which are elevated in plasma of Alzheimer patients) and association with CFH (Y402H) and APO E variation, etc.

Protein profiling studies of the drusen also revealed the presence of immunoglobulins, components of the complement pathway (C3, C5b-9 complex) (Johnson et al, 2000), molecules involved in acute phase response to inflammation (e.g. Amyloid P component and 1-antitrypsin), proteins that modulate immune response (vitronectin, clusterin, apolipoprotein E, membrane cofactor protein, complement receptor 1), major histocompatibility complex class II antigens and HLA-DR and cluster differentiation antigens (Mullins et al, 2000). Accumulation of all these proteins and cellular debris suggest that cells are subjected to a chronic sublethal complement attack mediated by choroidal dendritic cells and macrophages resulting in the further degradation of Bruch’s membrane (Johnson et al, 2001, Hageman et al, 2001). These studies gave a strong evidence for the role of inflammation and dysfunction of complement pathways in the pathogenesis of AMD.

ACCUMULATION OF LIPOFUSCIN IN THE RPE

Lipofuscin are autoflourescent lysosomal storage bodies that accumulate in the postmitotic metabolically active cells throughout the body due to normal aging process. These bodies originate primarily from the degradation of different intracellular organelle and the incomplete degradation of extracellular material taken into the cells by phagocytosis. RPE lipofuscin are formed due to ingestion of photoreceptor outer segments as evident from the presence of retinoids, high concentration of DHL-Lipids, etc. Accumulation of lipofuscin in RPE has been associated with the development of AMD though evidence for the same has not been provided.

ROLE OF OXIDATIVE STRESS MEDIATORS AND MODIFICATIONS IN AMD PATHOGENESIS

The evidence for oxidative stress playing a major role in AMD pathogenesis includes increased risk of AMD in smokers, presence of oxidative modified proteins in the drusen/Bruch’s membrane/ RPE / choriodal tissues and slower progression of AMD in patients fed on regular supplementation of antioxidant vitamins and Zinc. Several studies have pointed out that oxidative protein modifications serve as a primary catalyst for macular degeneration, the reactive oxygen species/oxidative products being provided by photo-oxidative rich milieu in the retina particularly the lipids and retinoid rich photoreceptor segments. Proteome study of drusens also demonstrated the elevated levels of oxidatively modified proteins like μ-carboxyethyl pyrrole (CEP) adducts in the drusen, Bruch’s membrane and plasma of AMD patients. It was found that the mean level of CEP adducts was 1.5 fold higher (p = 0.004) and that of antibody titers in plasma was 2.3-fold higher (p = 0.02) in patients with AMD compared with normal age-matched controls (Gu et al, 2003). Of individuals (n = 13) exhibiting both antigen and autoantibody levels above the mean for non-AMD controls, 92% had AMD. Based on the results, CEP immunoreactivity and autoantibody titer may have diagnostic utility in predicting AMD susceptibility.

These CEP adducts are generated from oxidation of polyunsaturated fatty acids (PUFAs) particularly docasohexanoic acid (DHA) present in photoreceptor outer segments and have been 9

Genetics of Age-related Macular Degeneration

10.Blander JM and Medzhitov R. Regulation of phagosome maturation by signals from toll-like receptors. Science 2004;304, 1014-18.

11.Bressler NM. Early detection and treatment of neovascular age-related macular degeneration. J Am Board Fam Pract 2002;15:142-52.

12.Burchard EG, Ziv E, Coyle N, Gomez SL, Tang H, et al. The importance of race and ethnic background in biomedical research and clinical practice. N Engl J Med 2003;348: 1170-5.

13.Cameron DJ, Yang Z, Gibbs D, Chen H, Kaminoh Y, et al. HTRA1 variant confers similar risks to geographic atrophy and neovascular age-related macular degeneration. Cell Cycle 2007;6:1122-5.

14.Castrillo A, Joseph SB, Vaidya SA, Haberland M, Fogelman AM, et al. Crosstalk between LXR and toll-like receptor signaling mediates bacterial and viral antagonism of cholesterol metabolism. Mol Cell 2003;12: 805-16.

15.Clark SJ, Higman VA, Mulloy B, Perkins SJ, Lea SM, et al. His-384 allotypic variant of factor H associated with agerelated macular degeneration has different heparin binding properties from the non-disease-associated form. J Biol Chem 2006;281:24713-20.

16.Conley YP, Jakobsdottir J, Mah T, Weeks DE, Klein R, et al. CFH, ELOVL4, PLEKHA1 and LOC387715 genes and susceptibility to age-related maculopathy: AREDS and CHS cohorts and meta-analyses. Hum Mol Genet 2006;15: 3206-18.

17.Cook DN, Pisetsky DS, Schwartz DA. Toll-like receptors in the pathogenesis of human disease. Nat Immunol 2004;5: 975-9.

18.Dandona L, Dandona R, Srinivas M, Giridhar P, Vilas K, et al. Blindness in the Indian State of Andhra Pradesh. Invest Ophthalmol Vis Sci 2001;42: 908-16.

19.De La Paz MA, Pericak-Vance MA, Lennon F, Haines JL, Seddon JM. Exclusion of TIMP3 as a candidate locus in agerelated macular degeneration. Invest Ophthalmol Vis Sci 1997;38: 1060–5.

20.DeWan A, Liu M, Hartman S, Zhang S, Liu DTL, et al. HTRA1 promoter polymorphism in wet age-related macular degeneration. Science 2006;314: 989-92.

21.Ebrahem Q, Renganathan K, Sears J, Vasanji A, Gu X, et al. Carboxyethylpyrrole oxidative protein modifications stimulate neovascularization: Implications for age-related macular degeneration. Proc Natl Acad Sci USA 2006;103:13480-4.

22.Edwards AO, Ritter R III, Abel KJ, Manning A, Panhuysen C, et al. Complement factor H polymorphism and agerelated macular degeneration. Science 2005;308: 421-4.

23.Fisher SA, Rivera A, Fritsche LG, Babadjanova G, Petrov S, et al. Assessment of the contribution of CFH and chromosome 10q26 AMD susceptibility loci in a Russian population isolate. Br J Ophthalmol 2006;91: 576-8.

24.Friedman E. The role of the atherosclerotic process in the pathogenesis of age-related macular degeneration. Am J Ophthalmol 2000;130: 658-63.

25.Gold B, Merriam JE, Zernanat J, Hancox LS, Taiber AJ, et al. Variation in factor B (BF) and complement component 2 (C2) genes is associated with age-related macular degeneration. Nat Genet 2006;38: 458-62.

26.Gotoh N, Yamada R, Hiratani H, Renault V, Kuroiwa S, et al. No association between complement factor H gene polymorphism and exudative age-related macular degeneration in Japanese. Hum Genet 2006;120:139-43.

27.Gu X, Meer SG, Miyagi M, Rayborn ME, Hollyfield JG, et al. Carboxyethylpyrrole protein adducts and autoantibodies, biomarkers for age-related macular degeneration. J Biol Chem 2003;278: 42027-35.

28.Haddad S, Chen AC, Santangelo SL, Seddon JM. The Genetics of Age-Related Macular Degeneration: A Review of Progress to Date. Surv Ophthalmol 2006;51:316-3.

29.Hageman GS, Anderson DH, Johnson LV, Hancox LS, Taiber AJ, et al. A common haplotype in the complement regulatory gene factor H (HF1/CFH) predisposes individuals to age-related macular degeneration. Proc Natl Acad Sci, USA 2005;102:7227-32.

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Textbook of Vitreoretinal Diseases and Surgery

Introduction

Cell therapy is an exciting area of research that is aimed at regeneration of tissues or organs in patients with irreparable damage to these tissues or organs. Cell therapy is no longer restricted to transfer of cells from a healthy subject to a patient, as in bone marrow transplant or corneal transplant. We can now envisage cell therapy that involves generation of patient-specific cells for transplantation, manipulate them in vitro into the desired cell type and transplant these cells into a patient for functional restoration of the damaged tissue. Although many cell therapies are currently being tested in animal models and their clinical application might take much longer, results from these studies are encouraging and suggest that regeneration of custom-made organs might indeed be possible in real life.

Cell therapy in the form of simple blood transfusion and bone marrow transplant has been in use for a long time. This was limited to collecting healthy cells from matched donors and introducing these cells into the patient. The cells were not propagated or manipulated in any way in vitro prior to transplantation. The first culture and transplantation of dermal epithelial cells was performed to replace damaged skin in burns patients1, 2 and taking cues from these experiments, similar approach was used to regenerate corneal epithelium in patients with limbal stem cell deficiency.3 Simultaneously embryonic stem cells were first established in 19814,5 and further understanding of mechanisms underlying self-renewal and differentiation of stem cells into various types of cells led to designing of new therapeutic strategies.

Degenerative diseases of retina like retinitis pigmentosa (RP), age-related macular degeneration (AMD) and diabetic retinopathy lead to irreparable damage to the retina and are a major cause of blindness in developed countries (Figure 2-1). Several genetic disorders such as retinitis pigmentosa or Leber congenital amaurosis also lead to retinal degeneration and blindness. A therapeutic approach that can regenerate retinal cells or the entire retina would help improve visual acuity and quality of life of these patients. Use of cell therapy for retina however is not easy as the retina consists of several cell types and retinal disorders can occur due to damage to any of these cell types. The neural retina consists of rod and cone photoreceptors, bipolar cells, ganglion cells, horizontal cells, Müller cells and amacrine cells that are arranged in a definite three-dimensional structure. The neural retina rests on the retinal pigment epithelium (RPE), which is in close contact with photoreceptors and is vital for maintenance of photoreceptor homeostasis. Degeneration of any type of cells in the neural retina or RPE can lead to retinal degeneration and loss of vision. Retinal disorders could also occur due to uncontrolled growth of endothelial cells leading to neovascularization in the retina that can eventually lead to retinal detachment in diseases like diabetic retinopathy and AMD. The current attempts at retinal cell therapy are therefore aimed at regeneration of cells in neural retina, regeneration of RPE and re-establishment of correct retinal vasculature. For treatment of inherited retinal disorders, the cell therapy also might involve manipulations to correct the genetic defect by gene therapy.

Cell therapy is an interdisciplinary field that requires inputs from the field of stem cell biology, regenerative biology, material science, developmental biology, genetic engineering and clinicians. The success and feasibility of any cell therapy depends on a number of factors, such as availability of suitable source of stem cells, expansion and manipulation of cells to generate the desired type of cell, introduction of cells in vivo, survival and integration of cells to reconstruct degenerated tissue and restoration of its physiological function (Figure 2-2).

An appropriate source of stem cells that can generate and sustain cells/tissue of interest is an 16 important starting point for cell therapy. Stem cells are the cells that have the ability to renew

Cell Therapy in Retinal Diseases

FIGURE 2-1: Fundus photograph showing wet (Top left) and geographic atrophy (Top right) age-related macular degeneration, and proliferative diabetic retinopathy (bottom)

themselves and also differentiate into any other cell of the body. They could be embryonic stem cells, which are derived from the inner cell mass of blastula or adult stem cells, which are present in the adult tissues such as bone marrow, liver, muscles, skin and cornea. Embryonic stem cells are totipotent, i.e. they have the ability to differentiate into any other cell type. Adult stem cells on the other hand have limited capacity to divide and can differentiate only into specific types of cells. They can be either multipotent stem cells that can differentiate into many types of cells, such as the bone marrow stem cells, or unipotent stem cells that can differentiate into only one type of cell, e.g. limbal stem cells. For retina, several sources of stem cells such as embryonic, fetal and adult stem cells from eye and bone marrow have been investigated as discussed below. An autologous source of cells would be preferred in order to avoid complications of rejection associated with graft rejection, but it may not be possible to use an autologous source if retinal degeneration is due to an inherited disorder.

The next important issue is the ability to culture these stem cells in vitro to expand them. The number of cells available for transplantation, especially if they are from adult tissues, is often limited and the cells need to be cultured in vitro to obtain sufficient number of cells. Expansion of cells while maintaining their characteristics such as their stemness or their differentiation status is a challenge

and requires standardization of culture conditions. This involves identification of correct culture 17