Molecular mechanism for choroidal neovascularization in age-related macular degeneration

Choroidal neovascularization (CNV) in age-related macular degeneration (AMD) is the most common cause of severe visual loss in patients over age 60 years in developed countries. While much is unknown about the underlying pathogenesis of CNV, the increased production of vascular endothelial growth factor(VEGF) by retinal pigment epithelium (RPE) is thought to play a central role in the development of this condition. However, recent studies using gene-manipulated mice question the importance of VEGF alone in promoting CNV. Angiogenesis is thought to result from the balance between angiogenesis stimulation and inhibition. A potent antiangiogenic factor recently has been identified in the retina and shown to be secreted by RPE cells. The inhibitor, pigment epithelium-derived factor(PEDF) is considered the key factor associated with avascularity of the cornea, vitreous, and outer retinal layer of the eye. We recently demonstrated that an imbalance between PEDF and VEGF in RPE cells caused by aging and oxidative stress may contribute to the disregulation of endothelial cell proliferation in CNV. In this review, we also discuss the angiogenic role of inflammatory cells in CNV, age-related changes in Bruch's membrane, and the possibility of the development of animal models reflecting CNV in AMD.     

Pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in aged human choroid and eyes with age-related macular degeneration

The purpose of this study was to examine the localization and relative levels of vascular endothelial growth factor (VEGF; an angiogenic factor) and pigment epithelium-derived factor (PEDF; an antiangiogenic factor) in aged human choroid and to determine if the localization or their relative levels changed in age-related macular degeneration (AMD). Ocular tissues were obtained from eight aged control donors (age range, 75-86 years; mean age, 79.8 years) with no evidence or history of chorioretinal disease and from 12 donors diagnosed with AMD (age range, 61-105 years; mean age, 83.9 years). Tissues were cryopreserved and streptavidin alkaline phosphatase immunohistochemistry was performed with rabbit polyclonal anti-human VEGF and rabbit polyclonal anti-human PEDF antibodies. Binding of the antibodies was blocked by preincubation of the antibody with an excess of recombinant human PEDF or VEGF peptide. Choroidal blood vessels were identified with mouse anti-human CD-34 antibody in adjacent tissue sections. Three independent observers graded the immunohistochemical reaction product. The most prominent sites of VEGF and PEDF localization in aged control choroid were RPE-Bruch's membrane-choriocapillaris complex including RPE basal lamina, intercapillary septa, and choroidal stroma. There was no significant difference in immunostaining intensity and localization of VEGF and PEDF in aged control choroids. The most intense VEGF immunoreactivity was observed in leukocytes within blood vessels. AMD choroid had a similar pattern and intensity of VEGF immunostaining to that observed in aged controls. However, PEDF immunoreactivity was significantly lower in RPE cells (p=0.0073), RPE basal lamina (p=0.0141), Bruch's membrane (p<0.0001), and choroidal stroma (p=0.0161) of AMD choroids. The most intense PEDF immunoreactivity was observed in disciform scars. Drusen and basal laminar deposits (BLDs) were positive for VEGF and PEDF. In aged control subjects, VEGF and PEDF immunostaining was the most intense in RPE-Bruch's membrane-choriocapillaris complex. In AMD, PEDF was significantly lower in RPE cells, RPE basal lamina, Bruch's membrane and choroidal stroma. These data suggest that a critical balance exists between PEDF and VEGF, and PEDF may counteract the angiogenic potential of VEGF. The decrease in PEDF may disrupt the balance and be permissive for the formation of choroidal neovascularization (CNV) in AMD.     

Expression of VEGF and PEDF in choroidal neovascular membranes following verteporfin photodynamic therapy

PURPOSE: To examine the impact of photodynamic therapy (PDT) on pigment epithelium derived factor (PEDF) expression in human choroidal neovascularization (CNV) membranes with regard to vascular endothelial growth factor (VEGF) expression. DESIGN: Interventional case series. METHODS: Retrospective review of interventional case series of 42 patients (42 eyes) who underwent removal of CNV. CNV was secondary to age-related macular degeneration (AMD) in all cases. Fifteen patients were treated with PDT, 3 to 246 days before surgery. CNV were stained for CD34, CD105, cytokeratin 18, VEGF, and PEDF. Twenty-seven CNV without previous treatment were used as control. RESULTS: Specimens without pretreatment disclosed varying degrees of vascularization, VEGF, and PEDF expression by different cells. Specimens treated by PDT, three days previously showed mostly occluded vessels lined with damaged endothelial cells (EC). In contrast, specimens excised at later time points after PDT were highly vascularized with healthy EC. This chronology was associated with an impressive VEGF immunoreactivity increased considerably in retinal pigment epithelial cells as well as significantly reduced PEDF expression in EC and stroma. CONCLUSIONS: PDT induces a selective vascular damage in CNV. The effectiveness of PDT, however, seems to be jeopardized by a rebound effect initiated by an enhanced VEGF and reduced PEDF expression in CNV.     

Inhibition of choroidal neovascularization by adenovirus-mediated delivery of short hairpin RNAs targeting VEGF as a potential therapy for AMD

PURPOSE: Choroidal neovascularization (CNV) is the leading cause of blindness in age-related macular degeneration (AMD). Several lines of evidence implicate increased levels of vascular endothelial growth factor (VEGF) in retinal pigment epithelium (RPE) from patients with AMD. Current approaches to attenuate VEGF or its receptors, including the use of small interfering (si)RNA, show significant promise, but still have limited efficacy and require repeat administrations, using procedures associated with multiple complications. The goal of this study was to develop an approach for long-term endogenous expression of short hairpin (sh)RNA that would significantly attenuate VEGF and hence act as a potential therapy for AMD. METHODS: Several shRNAs expressed from recombinant adenovirus were developed. These shRNAs were expressed in human RPE cells in the presence of adenovirus vectors overexpressing VEGF, and the amount of VEGF attenuation was evaluated. Adenovirus vectors expressing VEGF were subsequently injected into the subretinal space of mice, and induction of CNV was measured in the presence of adenovirus vectors expressing shRNA targeting VEGF. RESULTS: Potent shRNA sequences were identified that were able to silence VEGF in human RPE cells. When expressed from adenovirus backbones, these shRNA constructs silenced VEGF by 94% at a 1:5 molar ratio (VEGF to shRNA) and 64% at a 1:0.05 molar ratio. Adenovirus vectors expressing high levels of VEGF could induce CNV in mice within 5 days. Co-injection of VEGF-expressing viruses into mice with shRNA targeting VEGF led to a substantial (84%) reduction in CNV. CONCLUSIONS: shRNA targeting VEGF from adenovirus vectors allows potent attenuation of VEGF and prevents CNV. This approach shows promise as a therapy for AMD.     

Expression of pigment epithelium derived factor and vascular endothelial growth factor in choroidal neovascular membranes and polypoidal choroidal vasculopathy

AIMS: To determine whether pigment epithelium derived factor (PEDF), a protein that inhibits angiogenesis, is expressed in human choroidal neovascular membranes (CNVMs) and in tissues from an eye with polypoidal choroidal vasculopathy (PCV). In addition, to compare the expression of PEDF with that of vascular endothelial growth factor (VEGF), a known stimulator of angiogenesis, in these tissues. METHODS: CNVMs, associated with age related macular degeneration (AMD), angioid streaks, and PCV, were obtained during surgery. The expression of PEDF and VEGF in the excised subretinal fibrovascular membranes was determined by immunohistochemistry. RESULTS: PEDF and VEGF were strongly expressed in the vascular endothelial cells and retinal pigment epithelial (RPE) cells in the CNVMs where numerous new vessels were prominent (clinically active CNVMs). On the other hand, immunoreactivity for PEDF and VEGF was weak in the new vessels where fibrosis was prominent (clinically quiescent CNVMs). However, the RPE cells were still positive for PEDF and VEGF. The specimens from the eye with PCV also showed strong expression of PEDF and VEGF in the vascular endothelial cells and the RPE cells. CONCLUSION: Because PEDF is an inhibitor of ocular angiogenesis and an inhibitor of ocular cell proliferation, our results suggest that PEDF along with VEGF may modulate the formation of subfoveal fibrovascular membranes.     

Parallel findings in age-related macular degeneration and Alzheimer's disease

Age is a common risk factor for Alzheimer's disease (AD) and age-related macular degeneration (AMD). Because of the increasing age of the population, these two age-related diseases have recently received a great deal of attention. In addition to age as a risk factor, AD and AMD have many characteristics in common. An important characteristic common to both diseases is the presence of amyloid β (Aβ) in the senile plaques of the AD brain and in the drusen of AMD patients. We have focused on the role of Aβ as a key regulator of the progression from drusen to AMD, and our results have shown that Aβ causes an imbalance of angiogenesis-related factors in the retinal pigment epithelial (RPE) cells. Mice that lack the Aβ-degrading enzyme neprilysin develop RPE degeneration, and the sub-RPE deposits that are formed have features similar to those of AMD in humans. These data suggest that a common pathogenic mechanism might exist between AMD and AD. Thus, therapeutic approaches that have targeted Aβ in patients with AD can also be applied to AMD. In this review, we summarise recent findings on the shared characteristics and perspectives between AMD and AD, beginning with the mechanism of Aβ deposition and including a discussion of Aβ-targeted therapeutic approaches for both AD and AMD.     

Vitamin A up-regulates the expression of thrombospondin-1 and pigment epithelium-derived factor in retinal pigment epithelial cells

Vitamin A is essential for the visual system. It is metabolized in the retina and the resulting product, retinoic acid (RA), greatly affects the structure and functions of retinal pigment epithelial (RPE) cells. RPE cells produce a variety of extracellular matrix (ECM) proteins and angiogenic factors, both of which are expressed at varying levels in the normal RPE layer. In this study, we investigated the effect of all-trans-retinoic acid on the production of an ECM protein, thrombospondin-1 (TSP-1), and two angiogenic factors, pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) by RPE cells. RA increased the release of TSP-1 and PEDF, but not that of VEGF, from human RPE cells in vitro. In vitamin A-deficient mice, the expression of TSP-1 and PEDF in the RPE layer considerably decreased compared with that of normal control mice. The vitamin A deficiency hardly affected the accumulation of VEGF in the RPE layer. These findings suggest that vitamin A modulates the structure and anti-angiogenic functions of the RPE layer partly by up-regulating the expression of the angiogenesis-related ECM protein, TSP-1, and the anti-angiogenic factor, PEDF.     

Aqueous humor levels of vascular endothelial growth factor and pigment epithelium-derived factor in polypoidal choroidal vasculopathy and choroidal neovascularization

PURPOSE: To determine the aqueous levels of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) in patients with active polypoidal choroidal vasculopathy (PCV) and choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD) and pathologic myopia. DESIGN: Prospective, comparative control study. METHODS: Aqueous humors were collected from 32 eyes of 32 patients for either active PCV or CNV. Among them, 11 eyes had active and symptomatic PCV, 12 eyes had active CNV secondary to AMD, and nine eyes had active CNV of pathologic myopia. Levels of VEGF and PEDF were determined by commercially available enzyme-linked immunosorbent assay kits. A group of 10 aqueous samples from 10 patients who underwent cataract surgery without other ocular or systemic diseases comprised the controls. RESULTS: VEGF concentrations in aqueous humor were markedly increased in patients with PCV, CNV of AMD, and CNV of myopia when compared with the controls (analysis of variance [ANOVA], P < .001). VEGF levels in eyes with PCV were, however, significantly lower than those of exudative AMD (P = .045). The PEDF levels were also significantly different among the groups (ANOVA, P = .001), and we observed increased levels in PCV, CNV of AMD, and CNV of myopia. CONCLUSIONS: VEGF and PEDF factors were coexpressed and increased with positive correlation in aqueous humor of eyes with active PCV. The different levels of both factors in eyes of PCV and AMD might suggest distinct clinical entities or different angiogenesis courses between PCV and AMD.     

RAGE ligand upregulation of VEGF secretion in ARPE-19 cells

PURPOSE: The importance of VEGF in stimulating neovascular age-related macular degeneration (AMD) is well-recognized, but the initiating factors that induce local upregulation of VEGF remain unclear. The current study was conducted to test the hypothesis that activation of RAGE (receptor for advanced glycation end products [AGEs]) by its ligands, including AGEs, amyloid-beta peptide (Abeta), and S100B/calgranulins, some of which are known components of drusen and Bruch's membrane deposits, modulate secretion of VEGF by retinal pigment epithelial (RPE) cells. METHODS: ARPE-19 cells were used for all experiments. The cells were transfected with constructs encoding a signal transduction mutant of human RAGE to assess the RAGE-dependence of intracellular signaling. VEGF secretion and gene expression were assessed by ELISA and quantitative real-time PCR. SDS-PAGE and size exclusion chromatography were performed to analyze the structural changes of S100B after oxidation of its thiol groups under denaturing and nondenaturing conditions, respectively. NF-kappaB activation was assessed via electrophoretic mobility shift assay (EMSA). The impact of the NF-kappaB inhibition was assessed by using parthenolide. RESULTS: ARPE-19 cells basally secreted VEGF under normal cell culture conditions. Immobilized ligands of RAGE increased VEGF secretion in a RAGE-dependent manner. In contrast, soluble AGE-BSA, fresh Abeta, and S100B were less effective in increasing VEGF secretion. Studies with Abeta demonstrated that oligomeric and surface-immobilized forms of Abeta, but not soluble monomeric forms of Abeta, were effective upregulators of VEGF secretion via RAGE. Oxidation of S100B's thiol groups resulted in the formation of oligomers that displayed distinct RAGE biological activity compared with the simple dimeric form. RAGE-mediated upregulation of VEGF secretion by ARPE-19 cells was largely dependent on NF-kappaB, as indicated by studies with parthenolide. CONCLUSIONS: Immobilized or oligomerized ligands for RAGE induce RPE cells to increase VEGF secretion. NF-kappaB plays a central role in RAGE-dependent RPE secretion of VEGF. In AMD, activation of the RAGE axis in RPE cells may contribute to upregulation of VEGF, potentially inciting or propagating neovascular macular disease.     

老年性黄斑变性光学相干断层扫描观察

目的 观察萎缩型，渗出型老年黄斑变性（Age-related macular degeneration AMD）患者的光学相干断层扫描图像特征。比较光学相干断层扫描（Optical Coherence Tomography OCT）和荧光血管造影（Fluorescein angiography FFA）的特点，对脉络膜新生血管（Choroidal neovascularization CNV）进行OCT分型。方法 经FFA确诊的AMD57例76只眼进行OCT检查。结果 AMD患者色素上皮萎缩，软性玻璃膜疣，神经上皮和色素上皮脱离具有特有的OCT特征，OCT图像中视网膜神经上皮增厚、隆起反映视网膜下，视网膜层间积液。神经上皮或色素上皮（Retinal pigment epithelium，RPE）隆起，其下低反射区反映神经上皮或RPE层脱离。CNV的OCT图像分为边界清晰的CNV，边界模糊的CNV，纤维血管性RPE脱离。FFA中的典型性CNV相当于OCT图像中边界清晰CNV，隐匿性CNV相当于OCT图像中边界模糊CNV和纤维血管性RPE脱离。结论 OCT能特征性显示AMD中视网膜神经上皮隆起，视网膜层间积液，出血，神经上皮和RPE的脱离，且显示不同类型CNV的OCT特征。     

Magnetic nanoparticles conjugated with “RPE cell -MCP-1 antibody -VEGF antibody” compounds for the targeted therapy of age-related macular degeneration: a hypothesis

Age-related macular degeneration (AMD) is the leading cause of vision loss in the elderly throughout the world. Treatment of AMD utilizing retinal pigment epithelium (RPE) transplantation represents a promising therapy. However, simplex RPE transplantation can only replace the diseased RPE cells, but has no abilities to stop the development of AMD. It has been indicated that oxidization triggers the development of AMD by inducing the dysfunction and degeneration of RPE cells, which results in the upregulation of local monocyte chemotactic protein-1 (MCP-1) expression. MCP-1 induces macrophage recruiment which triggers local inflammation. As a result, the expression of vascular endothelial growth factor (VEGF) is upregulated by MCP-1 mediated inflammation and results in the formation of choroidal neovascularization (CNV). We accordingly propose a targeted therapy of AMD by subretinal transplanting the compound of RPE cell, MCP-1 antibody, and VEGF antibody and using a magnetic system to guide RPE cell compounds conjugated with superparamagnetic iron oxide nanoparticles (SPIONs). Furthermore, SPION-labelled RPE cells can be tracked and detected in vivo by non-invasive magnetic resonance imaging (MRI). This novel RPE cell transplantation methodology seems very promising to provide a new therapeutic approach for the treatment of AMD.     

Comparison of nonmydriatic digitized video fundus images with standard 35-mm slides to screen for and identify specific lesions of age-related macular degeneration

PURPOSE: The purpose of this study was to compare nonmydriatic digitized images obtained using a digital imaging system (resolution of 640 x 480 pixels) with 35-mm slide images for detecting specific findings of age-related macular degeneration (AMD) and to evaluate its usefulness as a screening tool in detecting signs of AMD. METHODS: Seventeen consecutive patients (33 eyes) underwent digital color imaging (with a nonmydriatic, 45-degree, fundus camera attached to a digital back) and standard 35-mm, 30-degree retinal color photography of the fundus: posterior pole, nasal retina, and temporal retina. The images were later reviewed for the presence or absence of specific retinal findings. The images were not compressed. Primary outcome measures included the presence or absence of drusen, hard exudate, choroidal neovascularization (CNV), subretinal hemorrhage, retinal pigment epithelial (RPE) changes, subretinal fibrosis, pigment epithelial detachment (PED), and subretinal fluid. Presence of drusen, with or without any one of the other findings, and presence of disciform scar or geographic atrophy were positive indications in screening for AMD. RESULTS: Agreement between image type was highest for PED (97%), CNV (91%), and subretinal fibrosis (91%); and lowest for RPE changes (63%). Sensitivity, specificity, positive predictive value, and negative predictive values were determined using the 35 mm-slide images as the reference for comparison. Sensitivity ranged from 40% (hard exudates) to 75% (subretinal hemorrhage). Specificity ranged from 88% (drusen) to 100% (hard exudate, CNV, RPE changes, PED). Positive predictive value ranged from 67% (subretinal hemorrhage) to 100% (hard exudate, CNV, RPE changes, PED). Negative predictive value ranged from 44% (drusen) to 97% (PED). For the purposes of screening for any evidence of AMD, the system was 70% sensitive. CONCLUSION: This digital fundus imaging system with 640 x 480 pixel resolution has low sensitivity and high specificity, as compared with 35-mm slide images, for detection of early AMD, but higher sensitivity for late findings (CNV, scar, atrophy) of AMD. Because of sensitivity for detecting any AMD coupled with the low sensitivity for detecting CNV, the system is not useful for evaluating AMD patients who require close follow-up and who are at risk for more severe visual loss.     

Expression of endostatin in human choroidal neovascular membranes secondary to age-related macular degeneration

Endostatin is an endogenous angiogenesis inhibitor which requires E-selectin for its antiangiogenic activity. The aim of this study was to investigate the expression of endostatin in human choroidal neovascular membranes (CNV) secondary to age-related macular degeneration (AMD) with regard to vascularization and proliferative activity. An interventional case series of 36 patients who underwent removal of CNV were retrospectively investigated. Thirty-six CNV were analyzed by light microscopic immunohistochemistry for the expression of CD34 (endothelial cells, EC), CD105 (activated EC), Ki-67 (cell proliferation), Cytokeratin 18 (epithelial cells), VEGF (vascular endothelial growth factor), E-selectin and endostatin. Donor eyes (n=7) including one with AMD were used as controls. Endostatin immunoreactivity was present in choroidal vessels of five as well as in the retinal pigment epithelium (RPE)-Bruch's membrane complex of two donor eyes without AMD. In one eye with AMD, endostatin was detected in RPE, Bruch's membrane and choroidal vessels. Ninety-two percent (33/36) of CNV disclosed endostatin staining. RPE-Bruch's membrane complex, choroidal vessels and stroma were positive in 50% (18/36), 72% (26/36), and 78% (28/36) of the membranes, respectively. Both control eyes and CNV expressed all the investigated markers except E-selectin being positive only in membranes. Endostatin, an endogenous angiogenesis inhibitor, is expressed in CNV and its therapeutic up-regulation may be a new strategy in the treatment of neovascular AMD.     

verteporfin光动力疗法对体外培养的成人视网膜色素上皮细胞色素上皮衍生因子、血管内皮生长因子表达水平的影响

目的 观察经光敏剂verteporfin光动力疗法（PDT）处理后的体外培养的成人视网膜色素上皮（RPE）细胞色素上皮衍生因子（PEDF）、血管内皮生长因子（VEGF）mRNA表达水平的改变。 方法 采用噻唑蓝比色法（MTT）测定PDT前后体外培养的成人RPE细胞活性的变化。应用半定量逆转录聚合酶链式反应(RT-PCR)测定PDT前后RPE细胞PEDF和VEGF mRNA表达水平的变化。 结果 PDT可造成RPE细胞死亡，死亡率与激光能量密度和光敏剂浓度成正相关。PDT后RPE细胞表达PEDF、VEGF mRNA水平下降，下降程度与激光能量密度和光敏剂浓度成正相关。 结论 PDT可下调体外培养的成人RPE细胞PEDF、VEGF mRNA的表达水平，这可能与PDT治疗黄斑下脉络膜新生血管膜（CNVM）后CNVM的消退或再生有关。 (中华眼底病杂志, 2006, 22: 256-260)     

Identification of <Emphasis Type="Italic">Chlamydia pneumoniae</Emphasis> within human choroidal neovascular membranes secondary to age-related macular degeneration

Age-related macular degeneration (AMD) is a leading cause of blindness in the United States, and increasing evidence suggests that it is an inflammatory disease. The prokaryotic obligate intracellular pathogen Chlamydia pneumoniae is emerging as a novel risk factor in cardiovascular disease, and recent sero-epidemiological data suggest that C. pneumoniae infection is also associated with AMD. In this study, we examined choroidal neovascular membrane (CNV) tissue from patients with neovascular AMD for the presence of C. pneumoniae and determined whether the pathogen can dysregulate the function of key cell types in ways that can cause neovascular AMD. Nine CNV removed from patients with neovascular AMD were examined for the presence of C. pneumoniae by immunohistochemistry (IHC) and polymerase chain reaction (PCR); in addition, we performed PCR on nine non-AMD eyes, and IHC on five non-AMD CNV, seven non-AMD eyes, and one internal limiting membrane specimen. Finally, human monocyte-derived macrophages and retinal pigment epithelial (RPE) cells were exposed to C. pneumoniae and assayed in vitro for the production of pro-angiogenic immunomodulators (VEGF, IL-8, and MCP-1). C. pneumoniae was detected in four of nine AMD CNV by IHC and two of nine AMD CNV by PCR, induced VEGF production by human macrophages, and increased production of IL-8 and MCP-1 by RPE cells. In contrast, none of the 22 non-AMD specimens showed evidence for C. pneumoniae. These data indicate that a pathogen capable of inducing chronic inflammation and pro-angiogenic cytokines can be detected in some AMD CNV, and suggest that infection may contribute to the pathogenesis of AMD. This study was presented in part at the 2003 and 2004 meetings of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, FL.     

吸烟对年龄相关性黄斑变性发生和发展的影响

年龄相关性黄斑变性(AMD)的视力损失多由视网膜色素上皮细胞的凋亡和光感受器的退化引起。主动、被动吸烟会增加AMD发病率及向晚期AMD进展的风险,并且影响湿性AMD的治疗效果。吸烟可引起脉络膜血管收缩、血管阻力增加、血管内皮功能障碍、脉络膜和神经节细胞复合体变薄,导致脉络膜和视网膜血管反应性受损。香烟中的尼古丁可导致血...     

Pigment epithelium-derived factor is deficient in the vitreous of patients with choroidal neovascularization due to age-related macular degeneration

PURPOSE: Pigment epithelium-derived growth factor (PEDF) is a potent inhibitor of angiogenesis that is found in the normal eye. The purpose of this study is to report decreased levels of PEDF in the vitreous of eyes with choroidal neovascularization (CNV) due to age-related macular degeneration (AMD). DESIGN: Prospective case-control study. METHODS: In a prospective case-control study, undiluted vitreous was collected from nine eyes of nine patients with CNV due to AMD and from an age-matched control group of 12 eyes of 12 patients with retinal disorders not involving neovascularization. Vitreous PEDF and vascular endothelial growth factor (VEGF) concentrations were determined by Western blot analyses and enzyme-linked immunosorbent assay (ELISA), respectively. Angiogenic activities of the vitreous samples were assessed in vitro using an endothelial cell chemotaxis assay. RESULTS: In vitreous samples from nine eyes with CNV due to AMD the mean +/- SD PEDF level was 2.8 ng/microl +/- 1.3 ng/microl. In vitreous samples from 12 age-matched control eyes the mean +/- SD PEDF level was 16.4 ng/microl +/- 7.1 ng/microl. The difference between the two groups was statistically significant (P =.00003). No significant difference in vitreous VEGF concentration was seen between CNV/AMD samples and control samples (P =.23). All CNV/AMD vitreous samples induced endothelial cell migration in vitro. No sample from age-matched non-age-related macular degeneration controls could induce endothelial cell migration, and 11 of 12 were able to block VEGF-induced migration in vitro. This inhibitory activity required active PEDF. CONCLUSION: The vitreous of patients with CNV due to AMD contained lower levels of PEDF and lacked the antiangiogenic activity of vitreous from age-matched controls. This suggests that loss of PEDF creates a permissive environment for CNV patients with AMD.     

Role of growth factors and the wound healing response in age-related macular degeneration

Growth factors (GF) are important in several stages of the pathogenesis of age-related macular disease (AMD). In choroidal neovascularization (CNV) in exudative AMD, the GF involved are similar to those involved in wound healing of the skin. Like granulation tissue of skin, CNV is characterized by clotting, inflammation, angiogenesis and fibrosis, and like in skin wounds, members of the VEGF, angiopoietin, PDGF and TGF- families of GF are expressed. However, several of these GF may also serve physiological functions in the normal eye, where the retinal pigment epithelium (RPE) employs them to provide trophic support to the neuroretina and choriocapillaris, in addition to maintaining an anti-angiogenic state. Derangement of these physiological functions may underlie the initiation of CNV in AMD. Basolateral secretion of VEGF-A by the RPE maintains the choriocapillaris, and is enhanced by hypoxia. Age-related changes in Bruchs membrane lead to impairment of this trophic function and choriocapillaris atrophy, as well as to decreased diffusion of oxygen towards the neuroretina. The resulting outer retina hypoxia may be an important driving force of CNV formation, by stimulating VEGF overexpression by the RPE, in addition to the effects of increased oxidative stress and low-grade inflammation. RPE senescence and hypoxia may also decrease expression of angiogenesis inhibitors such as PEDF, further shifting the balance to a pro-angiogenic state in the aging eye.