Ординатура / Офтальмология / Английские материалы / Ocular Disease Mechanisms and Management_Levin, Albert_2010

VEGF-A. Compared to bevacizumab it is smaller and has a greater affinity for VEGF.12 Two phase III clinical trials proved the efficacy of intravitreal ranibizumab as approximately 95% of patients lost less than 3 lines of vision in the treated groups versus nearly 65% in the controls. Perhaps even more surprising was that these trials represented the first prospective, randomized, controlled clinical trials to show a gain in visual acuity in neovascular AMD, with approximately 35% of treated patients gaining 3 or more lines of vision versus 5% in the control groups.13,14

Treatment of neovascular AMD is an area of blossoming growth. From initial treatment with thermal laser to modern approaches of VEGF blockade, the treatment of neovascular AMD is now aimed at early diagnosis and visual improvement rather than slowing the natural history. As we continue to understand more of the pathophysiology behind neovascular AMD, we will see a proliferation of new therapies and a combination of existing treatment options.

Epidemiology

AMD is a common disease that predominantly affects the elderly white population. Epidemiologic studies have provided estimates of disease prevalence in a variety of countries across the world. A large meta-analysis was performed providing pooled data on the prevalence of neovascular AMD by age, gender, and race.15 Selected data are presented in Table 69.1 and the reader is referred to the original studies for further details.

Diagnostic workup

The diagnosis of neovascular AMD is ascertained based on clinical symptoms and signs. Metamorphopsia and decreased visual acuity are the most common symptoms. Clinical examination by slit-lamp biomicroscopy may show a gray- ish-green membrane under the retina, hemorrhage under or within the retina, and/or RPE detachments. However, ancillary testing is an important adjunct to diagnosis in many cases and serves as confirmatory evidence prior to initiating treatment (Box 69.1).

Traditionally, ancillary testing and diagnostic confirmation were based on fluorescein angiography (Figure 69.2). With the development of optical coherence tomography

Figure 69.2  Fluorescein angiogram of a classic choroidal neovascular membrane.

Table 69.1  Prevalence rates for wet age-related macular degeneration

Modified from The Eye Diseases Prevalence Research Group. Prevalence of age-related macular degeneration in the United States. Arch Ophthalmol 2004;122:564–572.

Data are presented as prevalence per 100 individuals.

Box 69.1  Diagnostic workup for neovascular age-related macular degeneration includes many ancillary testing techniques

•Fluorescein angiography assesses leakage from incompetent neovascular vessels imaging the area of choroidal neovascularization

•Optical coherence tomography uses reflected light to create an optical cross-section of the retina enhancing morphometric evaluation

•Dynamic indocyanine green angiography assesses flow within the choroidal vasculature enabling visualization of the following vascular patterns:

Capillary-dominated lesions

Arteriolar-dominated lesions

Mixed capillary–arteriolar lesions

Figure 69.3  Optical coherence tomography of a patient with neovascular age-related macular degeneration showing evidence of both a pigment epithelial detachment and subretinal fluid.

Figure 69.4  Single frame from a dynamic indocyanine green angiography of a choroidal neovascular lesion demonstrating a mixed capillary–arteriolar lesion.

(OCT), this serves as a critical adjunctive tool (Figure 69.3). More recently, dynamic indocyanine green (ICG) angiography has allowed visualization of the neovascular complex and morphologic determination (Figure 69.4). Finally, the role of fundus autofluorescence is being evaluated for a complementary role in determining RPE health.16

Due to the biochemical properties of fluorescein dye, 15% is nonprotein-bound in the blood stream after injection. This allows fluorescein to leak out of incompetent neovascular vessels imaging the area of CNV. Fluorescein angiographic patterns can be subdivided into classic and occult based on the pattern of leakage. OCT has further helped diagnose and follow CNV lesions. In this imaging modality, reflected light is used to create an optical crosssection of the retina. Typical findings include any combination of the following: subretinal fluid, RPE detachments, diffuse retinal thickening, cystic changes within the retina, and hyperreflectivity corresponding to the neovascular lesion. Dynamic ICG angiography has been another area of recent development in defining morphologic patterns of CNV. Due to the near-infrared range of light absorption (~805 nm) and emission spectrum (~835 nm) and proteinbound affinity (98%) of ICG dye, visualization of the choroidal vasculature is possible. Furthermore, with the advent of confocal scanning lasers and rapid computer-processing algorithms, the sequence can be viewed as a dynamic movie (16 frames per second) versus static images (maximum 1 frame per second). This allows dynamic ICG to evaluate flow within the vasculature and not merely leakage, as traditional fluorescein and static ICG angiography have done in the past. The authors have evaluated hundreds of cases and determined the following patterns of pathophysiologic sig-

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Box 69.2  Subtypes of neovascular age-related

macular degeneration

•Traditional choroidal neovascularization is a process where new blood vessels grow from the choroid, breach Bruch’s membrane, and proliferate under the retinal pigment epithelium and/or retina

•Polypoidal choroidal neovascularization is defined as aneurysmal, or polypoidal, dilations of the inner choroidal vasculature

•Retinal angiomatous proliferation is defined as a neovascular process commencing with an angiomatous proliferation within the retina

nificance: capillary-dominated lesions, arteriolar-dominated lesions, and mixed subtypes of the two. Ongoing research into this modality will help to define the role of dynamic ICG angiography in prognosticating therapeutic responsiveness.

Differential diagnosis

The differential diagnosis of neovascular AMD can be viewed from three separate perspectives. First, as our understanding has developed surrounding neovascular AMD, three main subtypes have been delineated: polypoidal CNV, retinal angiomatous proliferation (RAP), and traditional CNV (Box 69.2).

Polypoidal CNV is defined as aneurysmal, or polypoidal, dilations of the inner choroidal vasculature and is best

imaged with ICG angiography.17 RAP is defined as a neovascular process commencing with an angiomatous proliferation within the retina that subsequently invades subretinally and eventually progresses to develop a chorioretinal anastomosis.18 Both polypoidal CNV and RAP are subtypes of the occult type of CNV and have been clinically detected using ICG angiography. Traditional CNV is a process where new blood vessels grow from the choroid, breach Bruch’s membrane, and proliferate under the RPE and/or retina.

The second perspective to view the differential diagnosis of neovascular AMD is other causes of CNV besides AMD. While the list is extensive, common diseases include high myopia, angioid streaks, choroidal rupture posttrauma, and intraocular inflammatory disease.

Finally, the differential diagnosis of neovascular AMD may be viewed from the perspective of other diseases that simulate CNV but are not true CNV. Diseases such as central serous chorioretinopathy and adult-onset foveovitelliform macular dystrophy can be difficult to distinguish from true CNV. To confound matters, both diseases can also be complicated by true CNV. However, typical history and ancillary testing will allow differentiation in challenging cases.

Pathology

The clinical hallmark of AMD in general is the presence of soft drusen and pigmentary changes in the macula. Pathologically this has correlated with basal laminar deposits. Basal laminar deposits represent material accumulation between the basal aspect of the RPE and its underlying basement membrane. As basal laminar deposits continue to enlarge, and eventually become continuous, a correlation with basal linear deposits is noted. Meanwhile, basal linear deposits represent accumulation of material between the basement membrane of the RPE and the inner collagenous zone of Bruch’s membrane. The combination of continuous basal laminar deposits and basal linear deposits represents the earliest pathological changes seen in AMD.19 The clinical manifestations of the advanced stages of theses pathologic changes are noted as drusen deposits under the neurosensory retina.

As AMD develops, morphologic changes in the macular region are noted. Eyes with neovascular AMD are noted to have increased degrees of calcification and fragmentation of Bruch’s membrane when compared to age-matched controls. These morphologic changes in Bruch’s membrane are not noted in subjects with nonneovascular AMD. However, the fellow eyes of subjects with unilateral neovascular AMD do have increased amounts of Bruch’s membrane calcification and fragmentation (Figure 69.5).20

Histopathology of excised choroidal neovascular membranes from human eyes with AMD has demonstrated a variety of structural and cellular components. Variability in the degree of fibrosis versus inflammatory nature of the membrane has been noted and may reflect activity and chronicity.21 Cellular components have included RPE cells, myofibroblasts, vascular endothelial cells, and inflammatory cells. In particular, while leukocytes were noted focally within the core of the membrane, macrophages were seen diffusely scattered. It is interesting to note that polymorphonuclear cells are rarely seen22 (Box 69.3).

Pathophysiology

Figure 69.5  Normal and pathologic changes associated with aging in neovascular age-related macular degeneration. RPE, retinal pigment epithelium; CNV, choroidal neovascularization; RAP, retinal angiomatous proliferation.

Box 69.3  Key pathologic features of neovascular

age-related macular degeneration

•Bruch’s membrane calcification

•Bruch’s membrane fragmentation

•Important cellular components of neovascular membranes

Inflammatory

•Macrophages: microglia and bone marrow-derived

•Leukocytes

Fibrosis

•Myofibroblasts

Other

•Vascular endothelial cells: local resident cells and vascular progenitor cell precursors

•Retinal pigment epithelium cells

Pathophysiology

A variety of etiologic components contribute to the pathophysiology of neovascular AMD. These include pathologic changes associated with aging, genetic polymorphisms, aberrant tissue response to injury from oxidative stress, and biochemical modulators of neovascularization (Figure 69.6). We will review each etiology and its accompanying pathophysiologic derangements in turn.

Pathologic changes associated with aging

As previously noted, neovascular AMD is accompanied by several morphologic changes in the choroid and Bruch’s membrane. Bruch’s membrane is actually a representation of several layers, including the basement membrane of the RPE, an inner collagenous zone, an elastic layer, an outer collagenous zone, and finally the basement membrane of the choriocapillaris. The compositions of basal laminar and basal linear deposits that accumulate in AMD speak to the mechanisms responsible for the development of this disease. Specifically, these deposits are comprised of lipids, proteins, cellular structures from RPE cells and choroidal dendritic cells, and a smaller amount of carbohydrates at their core.23 As proposed by Hageman et al,23 the biogenesis of drusen begins with RPE injury from a variety of factors, including genetic mutations and oxidative injury from light and systemic factors such as smoking, lipofuscin accumulation, and ischemia. This leads to RPE cell death and the release of stimulatory molecules (for example, cytokines) that recruit dendritic cells from the choroidal circulation. The dendritic cells send cellular processes through Bruch’s membrane and undergo local maturation to form the core of the drusen. Further amplification of this local inflammatory response may result in complement activation, extracellular matrix (ECM) proteolysis, and macrophage recruitment.

Other morphologic changes associated with aging include an age-related accumulation of lipofuscin in the RPE cytoplasm from 1% by volume in the first decade of life to 19% in the ninth decade, thickening of Bruch’s membrane from 2 m at birth to 4–6 m by the 10th decade of life, and decreased permeability of substances across Bruch’s mem- brane.24–26 It is likely that this combination of aging in conjunction with genetic predispositions, drusen accumulation, oxidative stress, and inflammatory changes contributes to the neovascularization seen in neovascular AMD.

Genetics

Genetics is a well-established risk factor for the development of AMD. Epidemiologic evidence in twin studies has demonstrated a higher concordance among monozygotic versus dizygotic twins.27 As genetic testing has evolved several genes have been isolated that portend a high risk for the develop-

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ment of AMD. In an excellent review by Montezuma et al28 the authors note the most commonly implicated gene is complement factor H (CFH) on chromosome 1q. Additionally, the genetic polymorphisms seen in the genes PLEKHA1/ LOC387715/HTRA1 on chromosome 10q appear to be important. The genetic polymorphisms associated with these genes account for, approximately, a 2–10-fold increased risk of developing AMD independent of, but modified by, other risk factors such as ethnicity, environmental exposures (e.g., smoking), and systemic health (e.g., body mass index). CFH gene polymorphisms may cause loss of inhibition of the complement system thus allowing complement-mediated damage of the RPE. Meanwhile, HTRA1 overexpression may disrupt Bruch’s membrane through the degradation of the ECM, which may permit vascular remodeling. Of the three proposed genes on chromosome 10q this is the most attractive based on biologic plausibility. Other genes identified in AMD linkage and association studies include adenosine triphosphate-binding cassette rim protein (ABCR), apolipoprotein E (ApoE), and specifically the e2 genotype, the HLA genes, and polymorphisms in the gene for vascular endothelial growth factor, to name a few.28

While these candidate genes have not all been evaluated in a population with only neovascular AMD, the genetic polymorphisms in CFH and LOC387715/HTRA1 have been assessed and shown to account for, on average, up to a fourfold and 33-fold increased risk of developing neovascular AMD, respectively.29

Response to injury

Injury to the RPE cells is believed to play a key role in the pathogenesis of neovascular AMD (Box 69.4). Injury comes from a variety of sources, including oxidative stress from the environment, systemic risk factors, and aberrant immunity. As noted above, injury to the RPE cells incites a cascade of events leading to drusen formation. Additionally, there is a strong inflammatory component to neovascular membranes (see Pathology). It is interesting to note the findings from the Age Related Eye Disease Study (AREDS)30 showing a statistically significant benefit (odds ratio 0.62, 99% confidence interval 0.43–0.90) in preventing the development of neovascular AMD in subjects at highest risk. This suggests a putative role of oxidative stress in the development of neo-

Box 69.4  Response to injury hypothesis

Enciting events

Oxidative stress

•Smoking

•Free radicals: generated from light exposure, retinal oxygen demands, and photopigment recycling

Accumulation of basal laminar and basal linear drusen

Effects on tissue

•Nonlethal cell membrane blebbing

•Dysregulated turnover of the extracellular matrix

•Inflammation

vascular AMD as the constituents of the vitamin supplementation under investigation are thought to exert their effect by acting as antioxidants. These findings highlight the importance of zinc and vitamins A, C, and E and epidemiologic data support the role of lutein, zeaxanthin, and omega-3 long-chain polyunsaturated fatty acids.31

Oxidative stressors

Oxidative stress via smoking is a well-established risk factor for AMD and may confer, on average, a twoto threefold risk of developing neovascular disease.32

Light exposure is another source of oxidative stress and may be involved in the production of free radicals. Free radicals are not merely produced due to the high irradiation levels of the retina but also due to the retina’s high oxygen demands and photopigment recycling with its incumbent induction of oxidative stress.33

The RPE and retinal tissue are also affected by basal laminar, basal linear, and clinically observable drusen. This abnormal accumulation of extracellular material under the RPE and within Bruch’s membrane leads to not merely a compositional change in the tissue but also to alteration in permeability to molecular flow to and from RPE cells.26 These findings imply a deranged tissue response that may contribute to further damage when under conditions of oxidative stress.

Effect of oxidative stress on tissues

While oxidative stress may lead to lethal responses, i.e., cell death, it may also lead to nonlethal responses that induce a functional change from baseline compatible with continued life but leading to tissue, and ultimately organ, dysfunction. The pathogenesis of this dysfunction can come from multiple etiologies, including cell membrane blebbing and dysregulated turnover of the ECM.

Nonlethal cell membrane blebbing is the process by which a cell can pinch off part of its plasma membrane and cytosol in an attempt to discard damaged cellular organelles, molecules, and lipid membranes. Importantly, nuclear fragmentation and cellular death do not occur.34

Dysregulated production and breakdown of the ECM may also contribute to the pathogenesis of AMD. This is because the normal anatomy and physiology of the ECM in most tissue require continuous turnover of collagen and other matrix components in a tightly regulated manner with rela-

Pathophysiology

tively small dysregulation producing profound changes in the ECM. This is evidenced by in vitro studies demonstrating RPE cellular dysfunction from oxidative stress and noting an abnormal increase in ECM with the accumulation of collagen and altered matrix metalloproteinase activity.35

Inflammatory response and tissue injury

The inflammatory response to oxidative injury is also an area of intense investigation. CNV membranes are known to have many cellular components, including inflammatory cells composed of a significant macrophage presence.21 It is interesting to note that macrophage depletion in animal models decreases the severity of CNV lesions.36 Additionally, patients with activated macrophages, defined as higher levels of tumor necrosis factor-α mRNA, demonstrated a fivefold increased prevalence of CNV secondary to AMD.37 Furthermore, many of the macrophages in CNV lesions have been shown to be composed of bone marrow-derived versus resident microglia (specialized tissue-resident macrophages within neuronal tissues), macrophages. This indicates a role of bone marrow-derived cellular constituents that may contribute to CNV.

Importance of extracellular matrix

The ECM is important to allow vascular remodeling. Specifically, the matrix metalloproteinases are a family of proteolytic enzymes that function to degrade the ECM. The tissue inhibitors of metalloproteinases serve to modulate this proteolytic activity and a pathological balance between the two is present in neovascular AMD.38

The inciting event is likely a combination of genetics, oxidative stress, and hypoxia and upregulation of proangiogenic molecules that ultimately leads to ECM breakdown, allowing vascular remodeling and neovascularization under the control of angiogenic mediators.

Neovascularization: angiogenesis and vasculogenesis

Changes in the local environment of the retina arise from a variety of etiologic sources, including genetics, aging, oxidative damage, and response to injury. Ongoing research indicates that these local changes combine to result in abnormal neovascularization typically arising from the choroid and growing toward the retina. As with all vascular growth, these vessels may arise as endothelial sprouts from pre-existing capillary channels (termed angiogenesis) or de novo from circulating bone marrow-derived cells (termed vasculogenesis) (Figure 69.7).39,40 It is helpful to look at each concept in turn to understand their contribution to what we have generically termed CNV in neovascular AMD.

Angiogenesis is an area of significant research. While many etiologic factors exist, the upregulation of angiogenic factors is at the heart of the concept and the RPE serves as an important modulator of this activity.41 In brief, proangiogenic molecules serve as a stimulus for the resident endothelial cells of the choriocapillaris to undergo proliferation.42 VEGF-A is known to be a key molecular component of this process. Yet, angiogenic molecules in general are

represented by proand antiangiogenic types. However these interactions are complex and some molecules regulate vascular proliferation differently in vivo compared to in vitro. In general, well-characterized proangiogenic molecules include VEGF and fibroblast growth factor with more complex roles for angiopoietins. Meanwhile, the bestcharacterized antiangiogenic molecule is pigment epithe- lium-derived growth factor.43 In the angiogenesis theory of neovascularization, proangiogenic factors regulate, in a multistep process, the proteolysis of the surrounding ECM, allowing endothelial cell migration, proliferation, and capillary tube formation.

In contrast, vasculogenesis refers to the formation of blood vessels de novo from circulating vascular progenitor cells (VPCs), which home to the site of future neovascularization and differentiate to become endothelial cells and vascular smooth-muscle cells (Box 69.5). The stimulus for recruitment of these cells may come from oxidative stress, hypoxia, or upregulation of angiogenic molecules, similar to the concept of angiogenesis.44 Yet, the distinguishing factor of the vasculogenesis theory is based on circulating VPCs rather than a simple proliferation of endogenous endothelial cells. Recent research into the role of vasculogenesis in CNV has begun to solidify this concept as a contributor to CNV in neovascular AMD.40,45 Specifically, identification of bone marrow-derived endothelial cells, vascular smoothmuscle cells, and macrophages from animal models of CNV has been demonstrated.46 Furthermore, approximately 50% of endothelial cells in neovascular membranes are noted to be derived from bone marrow sources, reiterating the importance of vasculogenesis in neovascularization.47

Conclusion

Understanding the pathophysiology of neovascular AMD is crucial to improving diagnosis and therapies for this disease.

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Box 69.5  Neovascularization: angiogenesis versus

vasculogenesis

•Angiogenesis is the development of new vessels from endothelial sprouts of pre-existing capillary channels

Key contributors

•Proangiogenic molecules: vascular endothelial growth factor and fibroblast growth factor

•Antiangiogenic molecules: pigment epithelium-derived growth factor

•Vasculogenesis is the development of new vessels de novo from circulating bone marrow-derived vascular progenitor cells

Key contributors

•Bone marrow-derived endothelial cells

•Bone marrow-derived smooth-muscle cells

•Bone marrow-derived macrophages

First, we need to refine our understanding of pathologic changes associated with aging and their relationship to genetic polymorphisms. Combined with tissue response to injury from oxidative stress and hypoxia we will unravel the association with angiogenic factor modulation. Finally, understanding the role of both angiogenesis and vasculogenesis in neovascular formation will allow further therapeutic intervention. By combining traditional examination techniques with imaging modalities such as dynamic ICG angiography we will be able to study in vivo these neovascular changes. With these pathophysiologic advancements new treatment modalities to control and finally cure this prominent disease will be developed.

6.Macular Photocoagulation Study Group. Visual outcome after laser photocoagulation for subfoveal choroidal neovascularization secondary to age-related macular degeneration. The influence of initial lesion size and initial visual acuity [see comment]. Arch Ophthalmol 1994;112:480–488.

7.Treatment of age-related macular degeneration with photodynamic therapy (TAP) study group. Photodynamic therapy of subfoveal choroidal neovascularization in age-related macular degeneration with verteporfin: one-year results of 2 randomized clinical trials – TAP report [see comment] [erratum appears in Arch Ophthalmol 2000;118:488]. Arch Ophthalmol 1999;117:1329–1345.

13.Brown DM, Kaiser PK, Michels M, et al. Ranibizumab versus verteporfin for neovascular age-related macular degeneration [see comment]. N Engl J Med 2006;355:1432–1444.

14.Rosenfeld PJ, Brown DM, Heier JS, et al. Ranibizumab for neovascular age-related macular degeneration [see comment]. N Engl J Med 2006;355:1419–1431.

15.Friedman DS, O’Colmain BJ, Munoz B, et al. Prevalence of age-related macular degeneration in the United States [see

comment]. Arch Ophthalmol 2004;122: 564–572.

17.Yannuzzi LA, Wong DW, Sforzolini BS, et al. Polypoidal choroidal vasculopathy and neovascularized age-related macular degeneration. Arch Ophthalmol 1999;117:1503–1510.

18.Yannuzzi LA, Negrao S, Iida T, et al. Retinal angiomatous proliferation in age-related macular degeneration [see comment]. Retina 2001;21:416–434.

23.Hageman GS, Luthert PJ, Victor Chong NH, et al. An integrated hypothesis that considers drusen as biomarkers of immune-mediated processes at the RPE–Bruch’s membrane interface in aging and age-related macular degeneration. Prog Retin Eye Res 2001;20:705–732.

24.Feeney-Burns L, Hilderbrand ES, Eldridge S. Aging human RPE: morphometric analysis of macular, equatorial, and peripheral cells. Invest Ophthalmol Vis Sci 1984;25:195–200.

29.Hughes AE, Orr N, Patterson C, et al. Neovascular age-related macular degeneration risk based on CFH, LOC387715/HTRA1, and smoking. PLoS Med 2007;4:e355.

30.Age-Related Eye Disease Study Research G. A randomized, placebo-controlled,

clinical trial of high-dose supplementation with vitamins C and E, beta carotene, and zinc for age-related macular degeneration and vision loss: AREDS report no. 8 [see comment]. Arch Ophthalmol 2001;119:1417– 1436.

37.Cousins SW, Espinosa-Heidmann DG, Csaky KG. Monocyte activation in patients with age-related macular degeneration: a biomarker of risk for choroidal neovascularization? Arch Ophthalmol 2004;122:1013–1018.

39.Killingsworth MC. Angiogenesis in early choroidal neovascularization secondary to age-related macular degeneration.

Graefes Arch Clin Exp Ophthalmol 1995;233:313–323.

45.Csaky KG, Baffi JZ, Byrnes GA, et al. Recruitment of marrow-derived endothelial cells to experimental choroidal neovascularization by local expression of vascular endothelial growth factor. Exp Eye Res 2004;78:1107–1116.

46.Espinosa-Heidmann DG, Caicedo A, Hernandez EP, et al. Bone marrowderived progenitor cells contribute to experimental choroidal neovascularization. Invest Ophthalmol Vis Sci 2003;44:4914–4919.

Clinical background

Ocular angiogenesis can be physiological or pathological, with physiological ocular angiogenesis occurring primarily during embryonic development (reviewed by Gariano1). Ocular angiogenesis in adults is usually pathological and is a major cause of vision loss and blindness due to conditions such as choroidal neovascularization (CNV) related to agerelated macular degeneration (AMD), diabetic retinopathy, neovascular glaucoma, corneal neovascularization, and retinopathy of prematurity. Each of these is discussed in at length elsewhere in this volume.

Research into mechanisms of physiological and pathological angiogenesis has led to a deeper understanding of molecular and cellular mechanisms involved in angiogenesis, with a principal focus of research efforts being the identification of molecules involved in promotion and inhibition of ocular neovascular disease. This chapter will focus on the molecules for which the role in ocular angiogenesis is best characterized, especially those that have led to the development of new drugs, and will present an overview of existing and developing therapies derived from this work.

Pathophysiology

The pathogenesis of ocular neovascularization involves a complex interaction between proangiogenic and antiangiogenic factors and molecules. There is also accumulating evidence supporting the inflammatory nature of both AMD and diabetic retinopathy. The depletion of monocytes inhibited pathologic (but not physiologic) retinal neovascularization in experimental models, strongly supporting a role for inflammation in ocular neovascular disease (Figure 70.1).2 Certain haplotypes of factor H, a regulatory component of the complement cascade, are associated with an increased risk of developing AMD (reviewed by Donoso et al3). In addition, complement factors C3a and C5a4 and C5b–9 (the membrane attack complex, or MAC)5 were identified in drusen of patients with AMD. Extensive deposits of C3d and C5b–9 have also been identified in the choriocapillaris of human eyes with clinically evident diabetic retinopathy, but not in the vast majority of control eyes,6 and elevated levels of assorted complement factors have been found in the vitreous of patients undergoing surgery for proliferative diabetic

retinopathy.7 Since complement is involved in opsonization, chemotaxis, and activation of leukocytes (reviewed by Rus et al8), the presence of complement in AMD and diabetic retinopathy lesions together with the co-localization of immune cells is evidence of active inflammation.

The final stage of angiogenesis involves stabilization of nascent vasculature by a process known as maturation, which involves selective pruning (remodeling) and recruitment of mural cells (pericytes and smooth-muscle cells). Leukocytes are believed to contribute actively to vascular pruning (Figure 70.2) through Fas/FasL-mediated endothelial cell apoptosis.9 Maturation is tightly regulated by levels of vascular endothelial growth factor (VEGF),10 with new vessels becoming refractory to VEGF withdrawal over time.11 In other studies, maturation corresponded with the expression of angiopoietin 1 (Ang1) and platelet-derived growth factor-B (PDGF-B)12 and prevention of mural cell binding to the endothelium by PDGF-B blockade resulted in disorganized retinal vasculature; normalization was restored by the administration of Ang1.13

The complexity of the pathogenesis of ocular neovascularization suggests numerous potential targets for intervention in the treatment of ocular neovascular diseases; yet this same complexity suggests that different interventions may be needed, either alone or in combination, to provide optimal benefits to all patients.

Endogenous promoters of angiogenesis

There are many molecules known to promote angiogenesis for which there is evidence supporting a role in the etiology of ocular neovascular disease (Box 70.1).

Vascular endothelial growth factor

A major research effort has identified VEGF as a master regulator in both physiologic and pathologic angiogenesis and a major contribution to ocular neovascular diseases. Elevated levels of VEGF have been shown to accompany the development of neovascularization in conditions such as retinal vein occlusion,14 neovascular glaucoma,15 retinopathy of prematurity,16 and proliferative diabetic retinopathy.14 VEGF has also been found to be overexpressed in the retinal pigment epithelium (RPE) in surgically excised CNV membranes of patients with AMD.17

A number of approaches have demonstrated that VEGF is both necessary and sufficient for the development of

Box 70.1  Promoters of angiogenesis relevant to

ocular neovascularization

•Angiopoietins

•Delta/Notch

•Ephrins

•Erythropoietin

•Fibroblast growth factor-2

•Integrins

•Matrix metalloproteinases

•Platelet-derived growth factor-B

•Tumor necrosis factor-α

•Vascular endothelial growth factor

ocular neovascularization. Experimentally induced ocular elevations of VEGF achieved by various means have led to pathological ocular neovascularization18–20 while inactivation of VEGF resulted in inhibition of ocular neovascularization.2,21,22 VEGF is produced by many cell types in the retina, including neurons, glia, and RPE cells.23–25 Hypoxia strongly induces the expression of VEGF through stabilization of hypoxia-inducible factor-1 alpha, a transcriptional regulator.26

Alternative splicing of the VEGF gene results in six major isoforms.27 Evidence from rodent models suggests that

VEGF164/165 (VEGF164 is the rodent equivalent of human VEGF165) was preferentially upregulated in ischemia-induced

pathological neovascularization and was significantly more potent at inducing inflammation.28

Figure 70.2  Vascular pruning is dependent on leukocyte adhesion. (A, B) Systemic administration of an antibody (Ab) against CD18, a molecule involved in leukocyte adhesion, reduced the number of adherent leukocytes in the normal rat retina on postnatal day 5, whereas a control antibody did not. (C) There was a significant reduction in the number of adherent leukocytes in rats treated with the anti-CD18 antibody

(n = 12; 59.9 ± 15.6) versus those treated with a control antibody (n = 10; 119.2 ± 21.9, P < 0.01). (D, E) CD18 blockade led to suppression of vascular pruning on postnatal day 6, when compared with those treated with a control antibody. (F) There was a significant reduction in the vascular density within five disc diameters of the optic disc between rats treated with the anti-CD18 antibody (n = 10; 36.7 ± 3.3%) and control rats (n = 8; 27.3 ± 2.7%, P < 0.01). (Modified from Ishida S, Yamashiro K, Usui T, et al. Leukocytes mediate retinal vascular remodeling during development and vaso-obliteration in disease. Nat Med 2003;9:781–788.)

Platelet-derived growth factor-B

The PDGF group of dimeric proteins is composed of combinations of four different polypeptide chains (PDGF A–D) with a cellular distribution that includes fibroblasts, vascular smooth-muscle cells, endothelial cells, RPE cells, and macrophages. PDGFs interact with two related tyrosine kinases, PDGF receptor (PDGFR)-α and PDGFR-β, leading to receptor dimerization and autophosphorylation (reviewed by Heldin and Westermark29).

During angiogenesis, the homodimeric PDGF-B has been found to play a particularly important role in the recruitment of PDGFR-β-expressing mural cells (pericytes and vascular smooth-muscle cells) to the developing vasculature (Figure 70.3).30 Jo et al11 employed three different murine models to study the contributions of signaling induced by VEGF and PDGF-B in ocular neovascularization. Physiologic development of neonatal retinal vasculature was significantly inhibited by blockade of signaling induced by PDGF-B but not by VEGF164; simultaneous blockade of both factors