Thesis
Reference
Le Ranibizumab Intravitreen induit une vasoconstriction artérielle rétinienne chez les patients présentant une dégénérence maculaire
liée à l'âge néovasculaire
PAPADOPOULOU, Domniki
Abstract
L'objectif de cette étude est d'analyser les effets secondaires des injections intravitréennes (IVT) de ranibizumab, inhibiteur de VEGF, sur le diamètre des artérioles rétiniennes chez des patients souffrant de dégénérescence maculaire liée à l'âge (DMLA) néovasculaire. Le diamètre des artérioles rétiniennes a été mesuré in vivo au moyen d'un analyseur de vaisseaux rétiniens (Retinal Vessel Analyzer-RVA) avant la première injection IVT, 7 et 30 jours après la première, deuxième et troisième injection, puis au 12 e mois de suivi. Une vasoconstriction significative des artérioles rétiniennes a été observée à la suite de chacune des trois premières injections IVT de ranibizumab. Elle a été permanente et significative par rapport aux valeurs mesurées à la baseline. Le ranibizumab IVT, mis à part l'inhibition de néovascularisation, provoque une vasoconstriction des artérioles rétiniennes dans les yeux atteints de DMLA néovasculaire, dont l'effet clinique n'est, à ce jour, toujours pas évalué.
PAPADOPOULOU, Domniki. Le Ranibizumab Intravitreen induit une vasoconstriction artérielle rétinienne chez les patients présentant une dégénérence maculaire liée à l'âge néovasculaire. Thèse de doctorat : Univ. Genève, 2010, no. Méd. 10624
URN : urn:nbn:ch:unige-129085
DOI : 10.13097/archive-ouverte/unige:12908
Available at:
http://archive-ouverte.unige.ch/unige:12908
Disclaimer: layout of this document may differ from the published version.
Section de médecine Clinique,
Département des Neurosciences cliniques et Dermatologie Service d’Ophtalmologie
Thèse préparée sous la direction du Professeur Constantin J. Pournaras
" LE RANIBIZUMAB INTRAVITREEN INDUIT UNE VASOCONSTRICTION ARTERIELLE RETINIENNE
CHEZ LES PATIENTS PRESENTANT UNE DEGENERESCENCE MACULAIRE LIEE A L’AGE
NEOVASCULAIRE "
Thèse
présentée à la Faculté de Médecine de l'Université de Genève
pour obtenir le grade de Docteur en médecine par
Domniki PAPADOPOULOU de
Thessaloniki, Grèce
Thèse n°10624
Genève 2010
Publications related to the thesis:
1. Papadopoulou DN, Mendrinos E, Mangioris G, Donati G, Pournaras CJ.
Intravitreal ranibizumab may induce retinal arteriolar vasoconstriction in patients with neovascular age-related macular degeneration.
Ophthalmology. 2009 Sep;116(9):1755-61. Epub 2009 Jun 27.
2. Mendrinos E, Mangioris G, Papadopoulou D, Donati G, Pournaras C.
One Year Results of the Effect of Intravitreal Ranibizumab on the Retinal Arteriolar Diameter in Patients with Neovascular Age-Related Macular Degeneration.
Invest Ophthalmol Vis Sci. 2009 Sep 24. [Epub ahead of print]
REMERCIEMENTS
Avant tout, j’exprime ma profonde gratitude au Professeur Constantin J. Pournaras sans qui, cette thèse de recherche en
rétine n’existerait pas, ainsi pour son soutien, ses qualités scientifiques et humaines incomparables.
Je tiens à remercier aussi le Professeur Avinoam B. Safran pour m’avoir offert l’opportunité de travailler dans un service de grande qualité, pour son ouverture d’esprit et son immense connaissance.
Un grand merci au Docteur Efstratios Mendrinos pour son soutien inconditionnel, son esprit critique, ainsi que pour son aide
précieuse à la composition de la présente thèse.
Je remercie vivement le Professeur Charles Riva pour son enthousiasme à m’apprendre tous les aspects scientifiques et
techniques de la recherche fondamentale.
Enfin, je remercie mon mari, George, pour son soutien et son amour, ainsi que mes enfants et mes parents qui m’ont aidée et
encouragée tout au long de ce travail.
INDEX
1. INTRODUCTION
2. PATHOGENESIS-THEORIES OF ETIOLOGY 2.1 Genetics
2.2 Hydrodynamic Changes 2.3 Hemodynamic Changes
2.3.1 Choroidal Blood Flow
2.3.2 Systemic Vascular Factors and AMD
2.3.3 Retinal Vascular Changes and Retinal Blood Flow
2.3.4 Anatomical Changes in CC/Bruch’s membrane/RPE in Dry AMD
2.3.5 Anatomical Changes in CC/Bruch’s membrane/RPE in Exudative AMD
2.3.6 Choroidal Watershet Zones and Neovascularization 2.3.7 Laser Doppler Flowmetry evaluation
2.3.8 Modifications of Vascular Blood Flow and Alterations Cascade 2.4 Vascular Endothelial Growth Factor (VEGF) agents
3. AGE-RELATED MACULAR DEGENERATION (AMD) 3.1 Ocular Manifestations
3.2 Non-neovascular AMD
3.2.1 Geographical Atrophy
3.2.2 Drusen and focal hyperpigmentation 3.3 Neovascular AMD
3.3.1 Retinal Pigment Epithelial Detachment (PED)
3.3.2 Fluoroscein angiography 3.3.3 Choroidal neovascularization 4. EPIDEMIOLOGY
5. TREATMENT
5.1 Treatment for non-exudative AMD 5.2 Low Vision rehabilitation
5.3 Laser Photocoagulation
5.4 PhotoDynamic Therapy (PDT) with Verteporfin 5.5 Transpupillary ThermoTherapy (TTT)
5.6 Anti-VEGF therapy 5.6.1 Pegaptanib 5.6.2 Ranibizumab 5.6.3 Bevacizumab 5.7 Corticosteroids
5.7.1 Triamcinolone
5.7.2 Combined intravitreal Triamcinolone and PhotoDynamic Therapy with Verteporfin
5.7.3 Anecortave 5.8 Surgical Approach
5.8.1 Submacular Surgery for Choroidal Neovascularization Membrane Excision
5.8.2 Macular Translocation 6. PURPOSE
7. MATERIALS AND METHODS 8. INJECTION TECHNIQUE
9. RETINAL ARTERIOLAR DIAMETER MEASUREMENT 10. EXAMINATION PROCEDURE
11. STATISTICAL ANALYSIS 12. RESULTS
13. DISCUSSION REFERENCES
Introduction
La dégénérescence maculaire liée à l'âge (DMLA) constitue la principale cause de perte visuelle dans les pays développés, et ceci malgré les importantes ressources consacrées à la recherche concernant sa prévention et son traitement. C’est une maladie avec un impact socio-économique significatif, sans traitement établi pratiquement jusqu’aux années quatre-vingts, et pour laquelle différentes modalités thérapeutiques ont été développées et testées jusqu’à nos jours, avec un succès variable. Des résultats particulièrement prometteurs pour la stabilisation, voire l’amélioration de la vision, ont dernièrement émergé grâce à l’introduction d’approches pharmacologiques contre l’angiogenèse, mais ces traitements sont relativement récents et leur efficacité et sécurité à long terme restent à définir.
La DMLA est une maladie fréquente de la macula, chronique, progressive et dégénérative, qui affecte des sujets âgés de plus de 50 ans et qui se caractérise par une diminution de la vision centrale. Au début du processus de la maladie, des lipids sont déposés dans la membrane de Bruch, probablement dûs au dysfonctionnement de l’EP pour traiter les débris cellulaires liés au turnover des segments externs des photoreceptor. L'aspect de drusen est le premiere signe clinique évident de la DMLA (Mullins R.F. et al 2000). L’analyse des drusen révèle qu’ils contiennent de la lipofuscine, de l’amyloïde, des facteurs du complément, et divers composants cellulaires (Gehrs K.M. et al 2006).En plus de drusen, dans la DMLA, on trouve un épaississement des couches collagènes de la membrane de Bruch, une dégradation de l'élastine et du collagène de la membrane de Bruch ainsi que sa calcification, des taux élévés des produits terminaux de la glycation, et l'accumulation des lipides et des protéines exogènes (Pauleikhoff D. et al 1990). Ces changements peuvent servir de barrière hydrophobe pour empêcher le passage de liquide et des nutriments entre la choroïde et la rétine externe ayant pour résultat une ischémie relative. Des foyers de neovascularization venant de la choriocapillaire peuvent alors se développer à travers de ruptures dans la membrane de Bruch. La maladie peut être classifiée en deux grandes catégories: non-néovasculaire (sèche) et néovasculaire (humide). Dans la DMLA sèche, la baissse d'acuité visuelle est la consequence de la parition des lesion atrophique. C’est un secteur ou ce sont des secteurs bien delimités d’
hypopigmentation ou de dépigmentation dûs à la degeneresence ou à la disparition des cellule de l’EP. Le néovascularisation dans la region de la macula est la caractéristique de la DMLA néovasculaire. La néovascularisation provident des
choriocapillaires sous la région maculaire ou peut surgir de façon predominante sous la rétine. Alors que la DMLA non-néovasculaire représente environ 80% de l'ensemble des cas diagnostiqués, la DMLA néovasculaire est responsable de près de 80% des forts handicaps visuels associés à cette maladie, compte tenue de la baisse considérable de l’AV quelle entraîne.
Les drusens peuvent être classifiées en dures et séreuses. Les druses dures sont des dépôts ronds, bien délimitées, jaunes-blancs, mesurant moins de 63 µm. Ces druses sont souvent identifiées dans différentes populations ; elles ne sont pas liées à l’âge et ne portent pas de risque élevé pour le développement d’une néovascularisation. En revanche, les druses séreuses sont mal délimitées, mesurant 63 µm ou plus ; elles sont liées à l’âge et associées au développement d’une néovascularisation. En cas de cette dernière, on observe une accumulation de liquide, une hémorragie et une exsudation lipidique à l’intérieur de la macula, qui peuvent aboutir à une fibrose, appelée cicatrice disciforme. D’autre part, en cas de forme sèche, grand nombre d’altérations anatomiques de la choriocapillaire, de la membrane de Bruch et de l’EP sont rencontrées. Quant au stade de l’atrophie géographique, les photorécepteurs et l’EP dégénèrent en fer à cheval arciforme entourant la fovéa ; la perte du réseau vasculaire choroïdien semble être un événement secondaire. [96]
L’atrophie géographique, la manifestation non-néovasculaire de la DMLA la plus grave, est à l’origine d'environ 21% des cas de cécité légale en Amérique du Nord.
(Leibowitz H.M. et al. 1973–1975)
Des changements anatomiques analogues de la choriocapillaire, de la membrane de Bruch et de l’EP sont observés dans la DMLA exsudative : Chez les patients avec une DMLA humide, caractérisée par la présence de liquide et/ou d’hémorragie sous l’EP ou sous la rétine neurosensorielle suite à une néovascularisation choroïdienne (NVC), une atténuation significative de la choriocapillaire autour d’une NVC active ou d’une cicatrice disciforme a été observée. Les lumières des choriocapillaires survivant et des néovaisseaux ont été toujours associées à des cellules d’EP viables, tandis que les régions sans EP comportaient des zones étendues de choriocapillaires viables diminués.
Les manifestations cliniques de la DMLA néovasculaire peuvent inclure les lésions suivantes : liquide sous-rétinien ; œdème maculaire ; hémorragie rétinienne, sous-rétinienne, ou sous l’EP ; exsudats lipidiques rétiniens ou sous-rétiniens ;
membrane en forme de plaque, ou décoloration grisâtre ou vert-jaunâtre bien délimitée ; décollement de l’EP ; déchirure de l’EP ; et fibrose sous-rétinienne ou cicatrice disciforme.
En ce qui concerne la DMLA sèche, une grande étude clinique multicentrique, à double insu, contrôlée versus placebo, connue sous le nom « AREDS » (Age- Related Eye Disease Study), a démontré qu’une formule spécifique d’antioxydants et de zinc à hautes doses diminuait significativement le risque de progression des les lésions précurseur (druse, altération de l’EP) et moindre le détérioration de fonction visuelle.
Les stratégies visant à traiter la NVC (néovascularisation choroïdienne) sont limitées par la localisation du tissu en prolifération, par la grande proximité entre ce tissu et de photoreceptor et l’EP facilement endommageables.
Différents traitements de la DMLA néovasculaire ont été soumis à une étude extensive au cours de grand nombre d’essais prospectifs randomisés. Ces traitements incluent la photocoagulation conventionnelle au laser, la photothérapie dynamique à la vertéporfine (Visudyne; Novartis Ophthalmics et QLT Inc.) et l’injection intra- vitréenne d’agents anti-VEGF incluant le pegaptanib sodique (Macugen;
Eyetech/OSI) et le ranibizumab (Lucentis; Genentech, South San Francisco). En outre, l’utilisation « off-label » du bevacizumab (Avastin; Genentech, South San Francisco) a connu une certaine popularité en tant que substance alternative au ranibizumab, grâce à son efficacité et sécurité évidentes, sa disponibilité universelle et son prix relativement bas.
Pendant grand nombre d’années, la photocoagulation au laser avait constitué le seul traitement établi contre la NVC qui était associée à une DMLA. La photocoagulation au laser est peu utilisée aujourd’hui pour traiter la DMLA néovasculaire. L’application de cette modalité thérapeutique est généralement limitée par la présence de critères d’éligibilité restrictifs, par la perte visuelle immédiate que celle-ci entraîne en raison du scotome induit par le laser, et par des taux de récidive particulièrement élevés. Ces inconvénients ont boosté la recherche vers la découverte de thérapies alternatives qui se caractériseraient d’une applicabilité plus étendue et
d’une efficacité plus élevée pour préserver, voire améliorer la vision et maintenir l’occlusion du réseau néovasculaire.
La photothérapie dynamique à la vertéporfine est un traitement efficace contre la NVC sous-fovéolaire liée à la forme néovasculaire de la DMLA. Elle consiste en l’injection systémique d’un médicament photosensible, suivie d’une irradiation de la membrane néovasculaire par une lumière non-thermique. Le médicament photosensible a une prédilection de se lier aux vaisseaux pathologiques. Lors d’une irradiation avec une lumière d’une longueur d’onde spécifique, la substance photosensible est activée, ce qui induit une réaction photochimique qui génère des radicaux libres sur la cible thérapeutique. Ces radicaux libres détruisent directement les cellules endothéliales et entraînent une adhésion plaquettaire secondaire massive, une dégranulation, une thrombose et une éventuelle occlusion des vaisseaux anormaux. [183, 184]
L’inhibition pharmacologique de l’angiogenèse est actuellement à l’étude au moyen d’acétate d’anécortave et d’autres agents spécifiques ciblant le facteur de croissance vasculaire endothélial (Vascular Endothelial Growth Factor-VEGF), un activateur connu de la NVC.
Alors que le VEGF existe sous plusieurs formes biologiquement actives, un fragment d’anticorps monoclonal humanisé recombinant (Fab), le ranibizumab, neutralise toutes les formes de ce facteur de croissance. En 2006, deux essais portant sur le ranibizumab ont montré que des injections intravitréennes mensuelles enrayaient la perte de vision et, dans de nombreux cas, amélioraient nettement l'acuité visuelle des patients atteints de DMLA néovasculaire. (Rosenfeld B.J. et al. 2006), (Brown D.M.
et al. 2006)
Le bevacizumab est un anticorps monoclonal pleine longueur qui, comme le ranibizumab, se lie à toutes les isoformes de VEGF et les inhibe, mais avec une affinité moindre, et possède une demi-vie plus longue par rapport au fragment d’anticorps.
(Brown D.M. et al. 2006)
Le 31 janvier 2006, l'Agence européenne des médicaments a accordé une autorisation de mise sur le marché au pegaptanib pour le traitement de la DMLA néovasculaire. Le pegaptanib est un oligonucléotide modifié pégylé qui se lie avec une haute spécificité et affinité au facteur de croissance vasculaire endothélial
extracellulaire (VEGF165) en inhibant son activité. Le VEGF165 est l’isoforme du VEGF impliquée préférentiellement dans la néovascularisation oculaire pathologique. Le pegaptanib bloque principalement le VEGF165, réduisant la croissance des vaisseaux sanguins anormaux ainsi que les saignements et les écoulements qui y sont associés.
Le VEGF peut cependant être considéré comme une arme à double tranchant.
Bien qu’il soit essentiel à la pathogenèse de la DMLA humide, il joue parallèlement un rôle capital dans le maintien de l’intégrité vasculaire, notamment dans des conditions d’ischémie et d’hypoxie. Ainsi, tous les effets bénéfiques des thérapies anti-VEGF appliquées à l’œil doivent être appréciés compte tenu des possibles effets systémiques à long terme de ces agents, sachant notamment que les puissantes thérapies «pan» anti- VEGF, telles que le ranibizumab et le bevacizumab, peuvent engendrer d’indésirables effets systémiques extra-oculaires dus au blocage des fonctions cardioprotectrices du VEGF.
L'objectif de cette thèse est d’étudier un certain nombre de questions se posant actuellement dans ce domaine: les propriétés et les fonctions du système VEGF, les effets des traitements VEGF et anti-VEGF sur les vaisseaux, en montrant que l’inhibition du VEGF peut avoir d’indésirables effets vasoconstricteurs, et les conclusions en matière d’efficacité et de sécurité du ranibizumab intravitréen dans le traitement de la DMLA humide.
Dans la présente étude, nous avons examiné l’effet à long terme du ranibizumab intravitréen (IVT) sur le diamètre des artérioles rétiniennes chez des patients atteints de DMLA néovasculaire au moyen d’un analyseur de vaisseaux rétiniens (Retinal Vessel Analyzer-RVA), un système de mesures objectives et reproductibles des changements de calibre des vaisseaux rétiniens.
L’étude a été réalisée conformément aux principes de la déclaration d’Helsinki.
Tous les sujets ont ainsi donné leur consentement éclairé avant de participer à l'étude.
La population étudiée comprenait 10 yeux appartenant à 6 hommes et 4 femmes, 4 yeux droits et 6 yeux gauches avec une moyenne d'âge de 77,5 ans (allant de 53 à 88 ans) au moment de l'étude.
Chaque patient a reçu au moins 3 injections mensuelles de ranibizumab au cours d’une période de 12 mois. Une moyenne de 4 injections (entre 3 et 10) avait été effectuée au 12e mois. Nous avons constaté une réduction significative du diamètre des artérioles rétiniennes à la suite de chacune des 3 injections IVT mensuelles de ranibizumab, avec une réduction moyenne de 18,6 ± 7,2 % un mois après la 3e
injection par rapport aux valeurs de référence. L’effet vasoconstricteur s’est maintenu jusqu’au 12e mois avec une réduction de 19,1 ± 8,3 % du diamètre des artérioles rétiniennes par rapport aux valeurs de référence après une moyenne de 4 injections.
Par ailleurs, cet effet vasoconstricteur semble être indépendant du nombre d’injections IVT de ranibizumab, aucune corrélation entre le nombre d’injections et le % de réduction du diamètre au 12e mois n’ayant été observée, peut-être cependant dû à la petite taille de notre échantillon. La PAM n’a pas subi de changement significatif au cours de la période de suivi, ce qui exclut un effet des fluctuations de pression artérielle systémique sur les diamètres des vaisseaux rétiniens mesurés. La PIO n’a pas subi de changement significatif non plus. Ni les facteurs systémiques, ni les facteurs locaux autres que le ranibizumab IVT n’ont donc probablement influé sur les mesures du diamètre des vaisseaux.
Nous présumons que les agents anti-VEGF mènent à la vasoconstriction artériolaire rétinienne directement, en inhibant la production du NO, et cet effet vasoconstrictif pourrait expliquer les modifications ischémiques rétiniennes qui ont été rapportés après leur administration intraoculaire. Cette étude constitue le premier rapport dans la littérature scientifique qui évalue l'effet à long terme d'un agent anti- VEGF sur le diamètre artériolaire rétinien. Nos résultats pourraient avoir des implications cliniques significatives, puisque la neutralisation à long terme du VEGF peut avoir des conséquences néfastes pour la rétine, i.e la perte des cellules neuronales de la rétine et de troubles circulatoires de la rétine et de la choriocapillaire.[273]
Nos conclusions suggèrent ainsi que le ranibizumab IVT, en diminuant le diamètre des artérioles rétiniennes, peut réduire le débit sanguin des capillaires rétiniens. D’autres études portant sur de plus larges échantillons sont nécessaires pour confirmer ces résultats ainsi que les éventuels effets négatifs sur la circulation rétinienne chez les patients atteints de DMLA néovasculaire et de maladies vasculaires rétiniennes.
1. Introduction
More than eight million Americans, particularly those over the age of 55 years, suffer from age-related macular degeneration, and the overall prevalence of advanced AMD is projected to increase by more than 50% by the year 2030. [1]
In the UK, the annual incidence of neovascular AMD was calculated to be around 24.000 in 2005, with prevalence of 243.000; this is predicted to rise to over 300.000 by 2025. [2]
The majority of patients with neovascular AMD progress to legal blindness in the affected eye within two years of diagnosis, and there is a 43% probability of progression to neovascular AMD in the other eye within five years. [1]
Healthcare utilization costs are seven times higher in affected patients compared to age-matched controls.
Until recently, the only pharmacological-based therapy for treatment of patients with neovascular degeneration has been photodynamic therapy with verteporfin. Thus, the development of new treatments for wet AMD, and of access to such treatments, was clearly important.
2. Pathogenesis-Theories of etiology
The causes of AMD and choroidal neovascularization are currently unknown.
One theory supports that abnormalities in the enzymatic activity of aged RPE cells lead to accumulation of metabolic by-products. Engorgement of RPE cells interferes with their normal cellular metabolism, leading to extracellular excretions [3, 4] In addition, lipids are deposited in Bruch's membrane, possibly because RPE fails to process cellular debris which are associated with outer segment turnover. The resulting hydrophobic barrier may prevent the passage of fluid from the retina to the choroid.
This causes the detachment of the RPE. Breaks in Bruch's membrane are considered to be responsible for neovascular ingrowth from the choriocapillaris.[5]
A more recent theory suggests that hemodynamic alteration in the choroidal circulation is an important pathophysiological mechanism.[6] The increased ocular rigidity and the decreased vascular compliance are the results of the atherosclerotic changes in the ocular vasculature. The result is the increased postcapillary resistance which leads to elevated hydrostatic pressure, with exudation of extracellular proteins
and lipids; these manifest as basal deposits and drusen. Corresponding degeneration of elastin and collagen causes calcification and fragmentation of Bruch's membrane. The increased levels of vascular endothelial growth factor (VEGF) are the result of the angiogenic stimulus that is caused by the relative choroidal ischemia.
This incites neovascular ingrowth from the choriocapillaris through a calcified, fractured Bruch's membrane. In support of this theory, Doppler imaging has confirmed choroidal vascular compromise in AMD patients relative to age-matched controls in several studies.[7-9]
Regardless of the mechanism of deposition, drusen are generally accepted to be precursor lesions for AMD when they are “soft” or “indistinct” (≥63µm). Small drusen (<63µm) are extremely common, with approximately 80% of the general population older than 30 years manifesting at least one. The number and confluence of drusen increase with age. After the age of 70 years, 26% of individuals have large or soft drusen, and 17% have confluent drusen.[10]
2.1 Genetics
Much evidence points to a familial component of AMD. Twin-concordance, linkage studies, and biomolecular investigations implicating genetic factors are compelling. Genetic analyses of AMD have been hindered by the late onset of the disease, which results in limited study pedigrees, as well as the intertwined nature of multiple genetic influences acting in concert with myriad environmental provocations.
Furthermore, the clinical classification of AMD may not lend itself to genetic analyses: clinical heterogeneity may not necessarily correlate with genetically different forms of AMD. For example, one study reported a significant odds ratio of 3.1 for exudative AMD in siblings, while the odds ratio of 1.5 for geographic atrophy was not statistically significant, suggesting varying relative genetic contributions for different subtypes of AMD.[11]
Nonetheless, familial aggregation of AMD has been demonstrated. First degree relatives of cases are three times more likely to develop exudative AMD than controls.[12]This study also estimated that the proportion of end-stage AMD in the population attributed to genetic factors was more than 20%. Twin studies suggest greater than 90% concordance in monozygotic twins.[13-15]Unlike these studies that used selected cases, a population-based study revealed an overall concordance of 37%
in monozygotic twins versus 19% for dizygotic twins for early AMD.179 This study estimated the overall inheritability of early AMD at 45%, with particularly high inheritability for 20 or more hard drusen (81%), large soft drusen (_125 µm) (57%), and pigmentary changes (46%).
Sibling analyses demonstrate that 55% to 57% of the variability in AMD could be attributed to a single gene segregation. (Heiba IM 1994)It is possible that a single gene is responsible for susceptibility, although many others could influence phenotype. In one large family with 10 AMD patients (predominantly with the dry phenotype) spanning three generations, linkage to chromosome 1q25-q31 has been reported.[16] A genome-wide scanning approach confirmed the presence of a susceptibility locus at 1q31 and identified another such region at 17q25.[17]
A statistically significant association of manganese superoxide dismutase gene polymorphism with AMD has been reported.[18]This is intriguing because of the role of this xenobiotic-metabolizing enzyme in oxidative stress, and highlights the interaction between environment and genetics in AMD. An allelic variant of the CST3 gene, which codes for cystatin C, an inhibitor of the cathepsin enzymes that function in the RPE to process rod outer segments, was associated with exudative AMD, particularly in men, in a German population-based study.[19] A specific Alu insert polymorphism, absent in the majority of the population, in the angiotensin converting enzyme (ACE) gene, which apart from its role in blood pressure regulation also plays a role in cell proliferation and death, seems to confer protection from the dry (atrophic) form of AMD.[20]
The apolipoprotein E ε4 allele is associated with a 57% reduction in the risk of exudative AMD, while the ε2 allele is associated with a 50% increased risk.[21, 22]
This finding assumes added interest in view of the presence of apolipoprotein E immunoreactivity in drusen and BlamD.[23]
Valuable information can be obtained from studying animal models of human disease. Over the last 25 years, various animal models have been developed displaying phenotypes of human AMD. Though no exact animal model of AMD exist, these models have enabled dramatic advances in our understanding of the potential molecular players in AMD and of therapeutic approaches to tackling the clinical progression of the disease. Epidemiological studies have provided researchers with environmental and genetic factors to manipulate and target in order to create potential animal models. An association between use of cholesterol lowering medications, such
as statins and reduced risk of early or late AMD has also been reported [24] These findings along with the identification of genetic loci such as different lipoprotein polymorphisms and risk of AMD[21, 22], have given rise to a series of models incorporating some form of a high fat diet. Apolipoprotein E3 (APOE3) Leiden transgenic mice express the human APOE3 Leiden gene and produce a dysfunctional form of human APOE3. When fed a high fat diet these mice develop basal laminar deposits, immunoreactive for human APOE[25]. Hypercholesterolemic mice produced either by APOE deficiency or by a high fat diet also develop thickened lipid-rich BrM with increased electron-lucent debris akin to that seen in human basal linear deposits [26, 27]. Interestingly, apolipoprotein E deficient mice exhibit accumulation of particles similar to BlinD[26] and BlamD[25] at an earlier age and have more membrane-bounded material in Bruch’s membrane than control mice.
Malek et al. reported an animal model with key features of dry AMD including diffuse and focal sub-RPE deposits, thickened Bruch’s membrane, and atrophy, hyper- and hypo-pigmentation of the RPE (Figure 1). Both choroidal and retinal neovascularization were also seen in a sub-population of these mice. This model is unique in that it was developed by combining three risks associated with AMD in a mouse; advanced age, genetic risk of apolipoprotein E isoform and environmental risk of a high fat and cholesterol diet. Interestingly, all three risk factors were necessary in order to produce an AMD-like phenotype, reflecting the complexity and multi-factorial nature of human AMD [28].
Figure 1: Morphology of APOE4 mice on HF-C diet, aged greater than 70 weeks, shows retinal pigment epithelial changes (RPE) such as RPE hyperpigmentation (A, arrow), hypopigmentation (B, arrow), diffuse (C) and focal sub-RPE deposits (D) an neovascularization (E). Images taken at 40 · magnification. Figure adapted from Malek G. et al. 2005. Copyright of Malek G. et al 2005
2.2 Hydrodynamic Changes
Retinal Pigment Epithelium is a melanin-containing epithelial layer that lies between the neural retina and choroid. The retinal pigment epithelium (RPE) is a vital tissue for the maintenance of photoreceptor function.[29]It is also affected by many diseases of the retina and choroid. Indeed, much of the pigmentary change that is visible clinically in retinal disorders takes place in the RPE (which is pigmented) rather than in the retina (which is transparent). Embryologically, the RPE is derived from the same neural tube tissue that forms the neural retina, but the cells differentiate into a transporting epithelium, the main functions of which are to metabolically insulate and support the overlying neural retina.
The RPE is a monolayer of cells that are cuboidal in cross section and hexagonal when viewed from above (Figure 2). The interlocking hexagonal cells are joined by tight junctions (zonulae occludens), which block the free passage of water and ions. This junctional barrier is the equivalent of the blood-retinal barrier formed by the capillary endothelium of the intrinsic retinal vasculature. All functions of RPE
are of great importance for the maintenance and metabolism of retina. RPE absorbs the scattered light, controls the fluid and nutrients in the subretinal space (blood- retinal barrier function), plays a pivotal role in visual pigment regeneration and synthesis and in synthesis of growth factors in order to modulate adjacent structures.
Moreover, RPE is responsible for the maintenance of the retinal adhesion, for phagocytosis and digestion of photoreceptor wastes, and for electrical homeostasis. In addition, RPE regenerates and repairs retina after injury or surgery.[30]
Figure 2: Anatomy and function of normal RPE.
In non-neovascular AMD, focal hyperpigmentation of the RPE is an important clinical feature. The risk of developing soft drusen and geographical atrophy increases in its presence. Visual acuity loss from dry AMD is generally due to geographic atrophy involving the foveal region. This is seen clinically as one or more well- delineated areas of hypopigmentation or depigmentation due to absence or severe attenuation of the underlying RPE. Alterations in the RPE consisting of focal hyper- or hypopigmentation are also associated with AMD, distinct from geographic atrophy.[31, 32]
Associated features of non-neovascular AMD such as drusen, focal pigmentary changes, and geographic atrophy are typically present in eyes manifesting
neovascularization (Figure 3). However, neovascularization secondary to AMD may occur without any of these precursor lesions; if they are not present, the neovascularization in older patients is most likely due to underlying AMD, but other possible causes must be considered. A retinal pigment epithelial detachment (PED) may be caused by serous fluid, fibrovascular tissue, hemorrhage, or the coalescence of drusen beneath the RPE. Each has a unique clinical appearance and exhibits specific patterns of fluorescence on angiography. Fibrovascular PED represents a type of occult CNV described later. Hemorrhagic PED manifests as a dark elevation of the RPE due to underlying blood, showing blocked fluorescence throughout all phases of angiography. Serous PED appears as a dome-shaped detachment of the RPE, exhibiting bright diffuse hyperfluorescence with progressive pooling in a fixed space.
Drusenoid PEDs, caused by coalescence of drusen, show staining, often with fading fluorescence in the late phase and an absence of leakage. [33]
Figure 3: Neovascular age-related macular degeneration. (A) The patient had choroidal
neovascularization followed by numerous episodes of hemorrhage, resulting in an organized scar. (B) A small vessel (C, capillary) has grown through Bruch’s membrane (B) into the sub-retinal pigment epithelial space, resulting in hemorrhage and fibroplasia. (C) The end stage shows a thick, fibrous scar between the choroid and the outer retinal layers (trichrome stain). Note the preservation of the retina, except for complete degeneration of the photoreceptors (B, Bruch’s membrane; C, choroid; NR, neural retina; S, sclera; ST, scar tissue). (B, Courtesy of WC Frayer; C, From Yanoff M, Fine BS. Ocular pathology, 5th ed. St. Louis: Mosby; 2002)
Bruch’s membrane is situated strategically between the nutrient fount of the choriocapillaris and the metabolically active RPE, which subserves photoreceptor homeostasis. As such, mechanical or functional injury to this two-way conduit for nutrients and cellular breakdown products would compromise retinal function. With age Bruch’s membrane undergoes numerous changes that impede normal filtration.
The progressive increase in lipid content in Bruch’s membrane throughout life is exaggerated in the macula compared to the periphery.[34]These neutral fat deposits differ from atheromas as they are derived from a cellular origin and not the peripheral blood, for they contain low levels of cholesterol ester and phosphotidylcholine, the major plasma lipids.[34-36] It is believed that progressive accumulation of extracellular material containing lipid in Bruch’s membrane may influence the development of AMD by altering its diffusion characteristics. The age-related exponential decline in hydraulic conductivity of Bruch’s membrane is more marked in the macula, and inversely related to its total lipid content. [37, 38] The impedance to diffusion through Bruch’s membrane compromises metabolic exchange between the choroid and retina,[37] and disrupts photoreceptor function.[39] Increased levels of
AGE[40] and RAGE[41] are found in AMD. The presence of AGE decreases the hydraulic conductivity of Bruch’s membrane.With age, Bruch’s membrane thickness increases (135% over 10 decades[42], thus increasing the diffusional path length. The accompanying 45% age-related decline in choriocapillaris density may further impair diffusion by reducing clearance from Bruch’s membrane.[42] A direct relationship between Bruch’s membrane thickness and RPE autofluorescence, a marker of lipofuscin accumulation, has been demonstrated in aged eyes.[43] Over 9 decades the permeability of Bruch’s membrane to serum proteins decreases by 90%.[44]
The solubility of collagen in Bruch’s membrane declines by 50–60% over 9 decades, and may contribute to debris accumulation in Bruch’s membrane.[45] With age, the glycosaminoglycan composition of Bruch’s membrane changes, increasing in size andin the total fraction of heparan sulfate, alterations that may compromise Bruch’s membrane filtration function.[46] Heparan sulfate also binds VEGF165, the predominant RPE VEGF isoform, and may contribute to choriocapillaris loss through the reduction of VEGF, an endothelial cell survival factor.[47]
It is believed that the origin of the abnormal deposits in Bruch’s membrane is from the RPE. Highly metabolic photoreceptors shed their outer segments that are then phagocytosed by the RPE and degraded by lysosomal enzymes. It is possible that the material discharged into Bruch’s membrane is abnormal as a consequence of incomplete phagolysosomal degradation, and in particular may contain large molecules and membrane complexes, obstructing diffusion. The activity of certain lysosomal enzymes is decreased within human RPE cells in aged eyes [48, 49] and various degradative enzymes have been identified with different regional distributions.[48]
This abnormal material may collect as discrete deposits in the inner portion of Bruch’s membrane between the basement membrane of the RPE and the inner collagenous layer (drusen). Additionally, diffuse thickening of the inner part of Bruch’s membrane (linear deposits) is seen with age. The similarity of staining pattern between debris accumulated in a diffuse and discrete manner implies a similar origin of linear deposits and drusen.
The accumulation of breakdown products derived from the RPE is believed to play a major role in the induction of RPE detachments and new vessel growth under Bruch’s membrane. Large confluent hypofluorescent, hydrophobic drusen predispose to RPE detachments, whereas hyperfluorescent, hydrophilic drusen predispose to
CNV.[50-52] This observation is in consonance with the suggestion that sub-RPE fluid is derived from the RPE,[53] wherein increased hydrophobicity of Bruch’s membrane would increase resistance to water flow from the RPE to the choroid, leading to RPE detachments. Conversely in the latter instance, greater exposure to serum derived angiogenic factors has been speculated to promote CNV.
2.3 Hemodynamic Changes
2.3.1 Choroidal blood flow: Choroidal blood flow is about 10 times higher than the flow in the grey matter of the brain and four times that of the kidney[54, 55], with flow estimates ranging from 500 to 2000 ml/min/100g [56-63], even though a corresponding difference in metabolic requirements does not exist.
This high rate of blood flow[64] is probably attributable to the low resistance of the choroidal vascular system, a consequence of the unusually large caliber of the choriocapillary lumen. Eighty-five of the total blood flow to the eye is distributed to the choroid and only 4% to the retina. The remaining flow perfuses the ciliary body (10%) and the iris (1%).[54, 57]
The more obvious function of high choroidal blood flow is the delivery of oxygen and nutrients and the removal of metabolic waste; the high blood flow would optimize the partial pressure and concentration gradients for efficient metabolic exchanges between the choroid and the retina. Consequently, the oxygen extraction from the choroidal blood is very low: the arteriovenous difference is only about 3%.
[57, 58] As a result of the low oxygen consumption from the choroïd, high tissue oxygen partial pressure (PO2) values (superior to 80 mmHg) are recorded at the choroïd.[65-67] High choroidal PO2 values are essential to assure about 65mmHg PO2 decrease between the choriocapillaries and the inner segments of photoreceptor.[68]
In pigs, about 60% of oxygen and 75% of glucose are delivered to the retina by the choroidal circulation, despite the low oxygen extraction from the choroidal blood.[69]
The oxygen content of the retinal venous blood in humans[70] and pigs [69] is about 38% lower than of arterial blood.
In man and other primates, the choroidal metabolic support of the retina is supplemented by the retinal vessels, present only in the superficial layers, and retinal blood flow is relatively small, i.e., 25 to 50 ml/min/100g.[57, 59, 60, 71, 72]
2.3.2 Systemic vascular factors and AMD: A great number of risk factors for the development of AMD are identical to them of cardiovascular pathologies. Systemic hypertension [73-75] history of coronary, carotid and peripheral vascular disease, [76]
serum cholesterol[77], dietary fat intake[78, 79], body mass index [80] and smoking[81, 82] are considered to be relevant risk factors. Among these potential risk factors, epidemiological studies have shown that only smoking has consistently been associated with AMD. [82]
As a consequence of these associations, patients of 49 to 73 years old presenting early clinical manifestations of AMD could be associated to higher risk of cerebrovascular strokes after 10 years follow-up.[83] Others added that AMD could be linked to higher risk of cardiovascular mortality.[84]
There have been reports linking statin use to a lower risk of AMD.[24, 85-87]
Several authors suggested that hypertension could increase the potential risk for the development of AMD based on the effects on choroidal circulation [88, 89]
Observational [74, 90] and prospective studies [73, 75] pointed out the association between hypertension and AMD risk. In the ‘Beaver Dam Eye Study’, the incidence of pigment abnormalities and the risk of AMD manifestation at 10 years was increased in the presence of high systolic blood pressure and pulse pressure at the initial examination[77]. A twofold to threefold risk of neovascular AMD in 10 years was associated with controlled or uncontrolled hypertension under medication at initial examination. A threefold risk of incident late stage AMD over the next 5 years was observed in people whose systolic blood pressure had increased more than 5 mmHg from baseline to the 5 year follow up examination, compared with people presenting stable systolic blood pressure values during the same period. The australian
‘Blue Mountains Study’ demonstrated that focal arteriolar narrowing was associated to the incidence of several AMD signs, [91] underlying the association with systemic hypertension.
As studies demonstrated an overlap of risk factors for AMD and cardiovascular disease, suggestion arises that a common disease mechanism may be operative in both AMD and cardiovascular disease and atherosclerosis, resulting in the deposition of lipid in the sclera and in Bruch’s membrane. The increasingly rigid sclera would act to encapsulate the ocular vasculature in a more incompressible compartment, leading to a greater degree of systolic-diastolic variation in the blood
velocity during the cardiac cycle.[1, 6, 92] This working hypothesis gives us the correlation between the alterations observed and systemic factors. Further investigations are needed to know whether AMD-related haemodynamic abnormalities are due to increased sclera rigidity, increased systemic vascular rigidity, or both.
2.3.3 Retinal vascular changes and retinal blood flow in AMD: Retinal vascular changes such as focal arteriolar narrowing, arterio-venous (AV) nicking, and generalized retinal arteriolar and venular narrowing were recently associated with cerebrovascular and cardiovascular outcomes such as stroke[93] and coronary heart disease,[94] and may be markers of microvascular damage from disease processes including prolonged hypertension and chronic inflammation[95]. Less is known regarding the associations of focal and generalized retinal arteriolar narrowing, AV nicking and retinopathy with AMD.
A limited number of population-based cohort studies have investigated the association of retinal vessel diameter and retinal vascular structural changes with the incidence of early (indistinct soft or reticular drusen or combined distinct soft drusen and retinal pigmentary abnormalities) and late (geographic atrophy or neovascularization) AMD.
In the Beaver Dam Eye Study, retinal vascular characteristics appeared to be weakly related and inconsistently associated with AMD.[73] Arteriole-to-venule ratio was only associated with the incidence of soft indistinct drusen and the incidence of RPE depigmentation. Focal retinal arteriolar narrowing was not associated with the incidence of AMD while AV nicking was associated with the incidence of early AMD. The Beaver Dam Eye Study also showed that retinal arteriolar diameter was not related to the 10-year incident early or late AMD and narrower arteriolar diameter was associated only with incident RPE depigmentation. [73]
Similarly, the Rotterdam Study found no association between incident AMD and retinal vessel (artery or vein) diameter.[96] This latter finding is consistent with data from the Atherosclerosis Risk in Communities Study [97] and the Blue Mountains Eye study.[91, 98]
In the Blue Mountains Eye study, focal arteriolar narrowing and AV nicking at baseline were weakly associated and of borderline significance with the 5-year
incidence of both early and late AMD.[91] In the Atherosclerosis Risk in Communities study, focal retinal arteriolar narrowing was associated with RPE depigmentation, although it was not associated with other AMD changes.[97]
In the 10-year incidence of early and late AMD and following adjustment for age, gender, smoking, and mean arterial blood pressure, the Blue Mountains Eye study found that the presence of focal arteriolar narrowing was associated with an increased risk of incident neovascular AMD and geographic atrophy, although only the latter reached statistical significance.[98] Focal arteriolar narrowing was not significantly associated with incident early AMD, whereas the presence of moderate/severe AV nicking at baseline remained significantly associated with incident early and late AMD. No significant association was found between baseline arteriolar or venular caliber and the 10-year incidence of late or early AMD, although a weak association, was observed between vessel calibers and pigment abnormalities.[98]
Taken together, these population-based data suggest that retinal arteriolar changes are inconsistently related to the incidence of AMD. Arterio-venous nicking seems to be more often associated with AMD than any other retinal vascular sign.
In stratified analyses, this association was found to be present in persons without hypertension, indicating that the AV nicking–AMD link may be independent of hypertensive processes.[98] On the contrary, there seems to be no association between the retinal vessel diameter and incident AMD. The significance and reproducibility of these findings is yet to be determined and deserve further research.
In recent years, a few studies have also investigated retinal blood flow in AMD.
Its relation to the disease has not been fully understood. Applying the laser Doppler technique to retinal arteries of eyes with various degrees of AMD, Sato et al found increasing pulsatility but constant blood flow, with increasing severity of AMD.[99]
This can be interpreted as an effect of reduced compliance proximal to the eye and not as increased distal vascular resistance, suggesting a more generalized systemic vascular pathology rather than an intraretinal vascular pathology. In another study, changes in retinal capillary blood flow in AMD were assessed with the Heidelberg Retinal Flowmeter.[100]These results indicated no change in blood flow in eyes with non-exudative AMD compared with healthy controls. However, reduced capillary blood flow in the disciform stage of late AMD and increased perimacular capillary blood flow was found in the exudative form of late AMD.[100] Such hemodynamic
data in late AMD need to be interpreted with caution as it is yet not clear whether abnormalities in retinal capillary blood flow are secondary to an autoregulative reaction or whether they are a primary mechanism in the pathogenesis of AMD.
Subsequent studies did not confirm the previous results and found reduced retinal capillary blood flow in eyes with non-exudative AMD [101]and no changes in retinal capillary blood flow in eyes with exudative AMD comparing to normal controls.[102]
More data about the changes in retinal blood flow in AMD are warranted.
2.3.4 Anatomical changes in choriocapillaris/Bruch's membrane/RPE in dry AMD: In geographic atrophy (GA), the photoreceptors and RPE degenerate in a horse shoe-shaped pattern surrounding the fovea; the loss of choroidal vasculature appears to be a secondary event.[103]
Quantification of CC (choriocapillaries) number and lumen diameters in cross sections, indicate a decrease with age and a further decline in both in AMD,[42] an increase in capillary density and a decrease in large blood vessel diameter [104] and a narrowing of CC lumen and loss of CC cellularity in AMD.[105]
In case of GA with areolar RPE atrophy, a severe 53% reduction in vascular density in the area from disk to the submacular region was observed, associated to an almost complete RPE atrophy. The border of the RPE defect was clearly delineated and coincided closely with the area of decreased choroidal vascular density. Surviving CC in the area of RPE atrophy had significantly smaller luminal diameters than CC in control subjects and in normal areas of the GA eyes. The study of GA subjects demonstrated that the RPE cells atrophied first followed by degeneration of the CC.[106] Therefore, in this form of AMD at least, the loss of choroidal vasculature appears to be a secondary event suggesting that GA is not of vascular etiology.
Interestingly, even in areas with complete RPE atrophy, some CC segments remained viable but severely constricted, contradicting the data found in animal models, suggesting that RPE are essential for survival of CC[107]
The association of surviving RPE cells with CNV in GA specimens suggest that RPE cells may furnish a stimulus for new vessel formation or stabilization.[108]
2.3.5 Anatomical changes in choriocapillaris/Bruch's membrane/RPE in exudative AMD: In subjects with wet AMD characterized by fluid and hemorrhage beneath the RPE or neurosensory retina due to choroidal neovascularization (CNV),
significant attenuation of CC around active CNV and disciform scars were observed.
The surviving CC and CNV lumens were always associated with viable RPE cells, and areas without RPE, had greatly reduced areas of viable CC as well.
According to the study of Lutty GA et all[109] it was apparent that RPE were present over areas with extremely attenuated CC, but the RPE were hypertrophic. In areas with active CNV, the CC was not viable and had extremely hypertrophic RPE covering the CNV, every example of active CNV was associated with surviving RPE[109]
2.3.6 Choroidal watershed zones and neovascularisation: In vivo studies have shown the choroidal vascular bed to be a segmental and an end-arterial system [110- 113] with each choriocapillaris lobule being a functional independent unit with no anastomoses with the adjacent lobules in the living eye.[111]
Observed areas of lobular ICG-dye filling were viewed as evidence that choriocapillaris blood flow can be at least functionally segmented.[114]
The choroid is therefore an end-arterial tissue and as such, has watershed zones located at the border between the areas of distribution of any two or more end- arteries. The apical parts of the various segments supplied by the short PCAs (Posterior Ciliary Arteries), meet each other in the center of the macula. Multiple watershed zones meet in the macula, and consequently are most vulnerable to ischemic disorders. Thus, the submacular choroid, is predisposed to chronic ischemia and neovascularisation more that any other part of the posterior choroid.
In patients with exudative AMD and choroidal watershed zone(s), three patterns were identified. The stellate pattern of the watershed zone was the most common one (60%), followed by the vertical (36%) and the angled one (4%).
Choroidal neovascularisation arose within the watershed zone in 88% of cases.[115]
Since the center of the watershed zone is the one that receives blood at lower pressure, it follows that a decrease in the perfusion pressure of the choroid causes a decrease in blood flow that is most severe in the center of the watershed zone. In the event of such a reduction in the choroidal perfusion pressure in association with the altered vascular regulation seen in patients with AMD [116], and/or with the AMD- related rarefaction and dysfunction of the choriocapillaris,[117] the resulting hypoxia- ischemia could disturb the physiologic balance between angiogenic and anti-
agiogenic factors predisposing to choroidal neovascularisation[118, 119] arising within or from the margin of the macular watershed zone(s).
2.3.7 Laser Doppler Flowmetry evaluation: Grunwald et al., using laser Doppler flowmetry, showed that there is a systematic decrease in choroidal circulatory parameters with an increase in the severity of AMD features associated with risk for the development of CNV, suggesting a role for ischemia in the development of CNV.[120]
2.3.8 Retrobulbar hemodynamic modifications: Friedman et al found reduced flow velocities and increased pulsatilities indices in the central retinal artery and short posterior ciliary arteries in a mixed group of patients with exudative and non- exudative AMD, as compared with healthy, age-matched control subjects, suggesting increased resistance of the choroidal vasculature. [7] Related to these results, they proposed a hemodynamic model of the pathogenesis of AMD and suggested that this disorder was caused by a progressive decrease in the compliance of the sclera and the choroidal vessels, leading to an increase in the resistance of the choroid to flow of blood.[1, 6, 92]
2.3.9 Modifications of vascular blood flow and alterations cascade: In the healthy or in AMD human eye, the choroidal blood flow evaluation and regulation experimentations allow a better understanding of the correlation between the pathological conditions and the choroidal circulation changes.
The results obtained by laser Doppler flowmeter indicate significant reduction of the choroidal blood flow correlated to the age and to the presence of AMD disease, compared to control subjects in the same range of age.
Although RPE metabolic modifications due to senescence processes are considered as the principal cause of the early clinical observations of AMD, choroidal vascular modifications seems to have secondary effects affecting the RPE photoreceptors interactions.
Resistance increase at the level of choroidal circulation consecutive to generalized vascular sclerosis [7, 92] and/or scleral rigidity increase,[121] associated
to blood flow decrease, modify the osmotic gradient through RPE leading to metabolic waste accumulation and drusen formation.
Choroidal circulation alterations may affect the metabolic exchanges through RPE and Bruch membrane, important to maintain visual function. Electrical activity of RPE cells being very sensitive to systemic hypoxia,[122] choroidal circulation alterations can affect oxygen and metabolic substrats diffusion through RPE and Bruch membrane, leading to functional alterations essential for vision due to dysfunction of the metabolic interactions between RPE cells and photoreceptors.
Blood flow alteration at the level of retrofoveal choroidal circulation contributes to the process of neovascularization characteristic of AMD. The fact that patients with high risk of neovascularization present the most severe decrease of choroidal blood flow underline the role of choroidal ischemia in the development of neovessels in the evolution of AMD.[120] Pathophysiological mechanisms for the development of subretinal neovascular tissue implicate release of neovascular mediators, potentially related to metabolic changes at the level of the external layers of the retina secondary to the decrease of choroidal blood flow.
Anatomical alterations linked to AMD (thickening of Bruch’s membrane, drusen formation) increase diffusion distance between choriocapillaries and photoreceptors internal segments. These anatomical alterations associated to the reduction of choroidal blood flow should enhance tissue hypoxia at the area of the photoreceptors inner segments, essential stimulus of vasoproliferative factors which are upregulated by hypoxia.[123, 124]
Neovascularization in the eye is also considered to result from an imbalance between stimulatory and inhibitory angiogenic factors.[123-125] Vascular endothelial growth factor (VEGF) is a likely candidate for the angiogenic stimulus for CNV as VEGF is produced also by RPE [126] and so it has provided a therapeutic target for CNV [127, 128]
The balance between VEGF and pigment epithelial growth factor (PEDF) in aging and AMD had been suggested that could shift the angiogenic potential in retina.[125, 129] PEDF was purified from the conditioned media of human retinal pigment epithelial cells and was found to be a potent inhibitor of angiogenesis [130]
and a neurotrophic factor. [131] PEDF administered intravitreally inhibits aberrant blood vessel growth in VEGF-induced neovascularisation.[132]
In eyes with AMD, PEDF levels were found significantly reduced in RPE cells, Bruch's membrane and choroidal stroma in contrast to VEGF immunoreactivity which was not significantly reduced in the RPE/Bruch's membrane/CC complex.[133]
Several of the anti-angiogenic agents as endostatin, which is the proteolytically cleaved carboxyl terminus globular domain of collagen XVIII (coll XVIII) [134] or thrombospondin-1 (TSP-1), produced by RPE in culture [135, 136]
detected in vitreous and aqueous humor,[137] are potentially involved in AMD.
Endostatin inhibits endothelial cell proliferation and migration in vitro and potently inhibits angiogenesis in vivo,[138] inhibiting the mitogen-activated protein kinase (MAPK) activation in endothelial cells.[134] Endostatin is significantly reduced in CC, Bruch’s membrane, and RPE basal lamina in AMD compared with aged control choroids. [139] TSP-1 in Bruch's membrane declines with age and it is almost absent in AMD, being significantly reduced in Bruch’s membrane, CC, and walls of large choroidal blood vessels.[140] The decline in the endogenous anti- angiogenic substances potentially favours the development of CNV.
The contribution of the vascular disturbances in the pathogenesis of AMD is actually established. However their precise role is not determined yet; associated to multiple factors affecting the metabolism of the RPE and/or genetic factors, lead to the appearance of the various phenotypes of AMD.
2.4 Vascular Endothelial Growth Factor (VEGF) agents
The development of new capillaries from preexisting networks, angiogenesis, is integral to embryonic development, somatic growth, and tissue repair. However, it also can prove harmful by feeding tumor growth or by destroying normal ocular architecture via aberrant neovascularization. An assemblage of molecular players orchestrates the complex process of vessel formation, and their identification is largely the serendipitous consequence of oncology research.
It is clear that vascular endothelial growth factor (VEGF) plays an important role in promotion of the neovascularisation and vessel leakage that lead to loss of central vision. VEGF was first described as a tumour-derived factor with potent ability to induce endothelial cell permeability,[141]proliferation and angiogenesis.[142, 143]
VEGF induces angiogenesis, and increases vascular permeability and inflammation: all
of these are thought to contribute to the progression of the neovascular form of AMD.
VEGF levels are raised in the retinal pigment epithelium and choroidal blood vessels of the macula and in the ocular fluid of most patients with proliferative diabetic retinopathy and retinal vein occlusion.[141-143]
However, VEGF plays a pivotal role in maintaining vascular integrity, particularly under conditions of ischaemia and hypoxia. Anti-VEGF agents administered systematically for other indications in oncology have been associated with serious systemic adverse events and death. [144]
Pharmacological inhibition of angiogenesis is currently being studied using anecortave acetate and other specific agents that target VEGF, a known promoter of CNV.[29]
VEGF, may be regarded as a double-edged sword. Thus, any beneficial effects of anti-VEGF therapies in the eye must be weighed against potential long-term systemic effects of these agents, particularly when potent [145]-antiVEGF therapies, such as ranibizumab and bevacizumab, may exert unwanted systemic extra-ocular effects due to the blocking of the cardioprotective functions of VEGF.
Hence, reduced endothelial Nitric Oxide (NO) release may accelerate the progression of atherosclerotic lesions. Most importantly, NO is the downstream mediator of VEGF and is considered an important defence system in maintaining vascular integrity.[146]
Up to 2003 there was no data on the role of NO in the regulation of human retinal blood flow available. Dorner et al.,[147] made a study in order to elucidate the role of NO in the maintenance of basal vascular retinal tone in humans. In addition, they investigated the possible role of NO in the hyperemic response to flickering light in the retina. Retinal arterial and venous diameters were therefore compared during placebo infusion and during infusion of NG-monomethyl-L-arginine (LNMMA), a competitive inhibitor of NO synthase. This was done during resting conditions as well as during flicker periods. They have shown that NO has an important role in the control of basal retinal vascular tone as well as in flicker-induced retinal vasodilation in humans.
It is of note that VEGF, mostly through its down stream mediator NO, has many essential physiological functions in maintaining vascular integrity, including the potential formation of collateral vessels crucial for the maintenance of perfusion to ischaemic tissues, as in acute myocardial infraction, in particular.[148]
Non-selective “pan”-anti-VEGF antagonism with ranibizumab or bevacizumab could be of even greater concern than blocking VEGF with selective antagonists such as pegaptanib. However, whether and to what degree more selective VEGF inhibition translates into fewer unwanted systemic effects remains unproven.
The aim of the thesis was to investigate some of the current issues in this field:
The properties and functions of the VEGF system, the effects of VEGF and anti-VEGF treatments on the vasculature, indicating that VEGF inhibition could have vasoconstrictive unwanted effects and findings on efficacy and safety of intravitreal ranibizumab in the treatment of wet AMD.
3. Age-related Macular Degeneration
Age-related macular degeneration (AMD) is the leading cause of central visual loss among individuals 65 years of age and older in developed countries.[4, 149-151]
The disease primarily affects the choriocapillaris, Bruch's membrane, and retinal pigment epithelium (RPE). However, the photoreceptor dysfunction due to underlying atrophy or choroidal neovascularization (CNV), with its corresponding fluid accumulation, hemorrhage, lipid exudation and fibrosis, typically lead to the visual loss.[3] The cause of the disease remains unclear, the treatment is unsatisfactory till now, and the prevention is not possible in the majority of the cases, despite the extensive research trials.
We can classify AMD in two categories: non-neovascular (dry) and neovascular (wet). Approximately, the 80% of all cases of AMD belong to the non-neovascular type of the disease. Nevertheless, neovascular AMD is responsible for nearly 80% of significant visual disability associated with this disease. The geographical atrophy is the most severe non-neovascular manifestation of AMD and it causes approximately 21%
of the cases of legal blindness in North America.[150]
3.1 Ocular manifestations
The clinical symptoms of the disease can be either metamorphopsia or blurred vision or lack of all these symptoms. Reported symptoms were also the decreased ability to read text in dim light and difficulty with dark adaptation. The most common way of beginning is subacute, except in some cases of neovascular AMD in which
