Expression of connective tissue growth factor and its potential role in choroidal neovascularization

BACKGROUND: To determine the expression of connective tissue growth factor (CTGF) in choroidal neovascularization. METHODS: Surgically excised choroidal neovascular membranes (CNVMs) were obtained at vitrectomy from eight eyes with age-related macular degeneration, five eyes with high myopia, and two eyes with angioid streaks. Light microscopic immunohistochemical analysis was performed to detect CTGF, transforming growth factor beta 1 (TGF-beta1), vascular endothelial growth factor (VEGF), pancytokeratin, and smooth muscle actin (SMA). RESULTS: CNVMs were classified by fibrotic status as cellular CNVM, moderate fibrous CNVM, and extensive fibrous CNVM. CTGF expression was found in vascular cells, stromal cells, and retinal pigment epithelium (RPE) cells. For the stromal cells, fibroblastlike cells were most strongly positive for CTGF. CTGF immunoreactivity in the stroma was stronger in the fibrous CNVMs than in the cellular CNVMs. Immunohistochemical analysis of serial sections revealed colocalization of CTGF with TGF-beta1 and VEGF; colocalization of CTGF with pancytokeratin and SMA was also found. CONCLUSION: Our findings suggest that transdifferentiated RPE cells and vascular cells possess remarkable CTGF expression in CNVMs. This expression of CTGF may stimulate fibroblasts to produce extracellular matrix and may promote angiogenesis in vascular cells. Colocalized TGF-beta1 and VEGF may also contribute the upregulation of CTGF.     

Expression of pigment epithelium-derived factor in normal adult rat eye and experimental choroidal neovascularization

PURPOSE: Pigment epithelium-derived factor (PEDF) is a protein produced by the retinal pigment epithelial (RPE) cells. Recent studies have implicated PEDF in activities that are inhibitory to angiogenesis. In this study, the expression of PEDF was investigated in normal rat eyes and in eyes with experimentally induced choroidal neovascularization and compared with the expression of vascular endothelial growth factor (VEGF). METHODS: Choroidal neovascularization was induced by laser photocoagulation in rat eyes. At intervals of up to 2 weeks after photocoagulation, the eyes were removed and prepared for in situ hybridization and immunohistochemical study. In situ hybridization was performed with digoxigenin-labeled PEDF riboprobes. Protein expression of PEDF and VEGF was studied immunohistochemically. RESULTS: In normal adult rat eyes, PEDF mRNA was observed mainly in the corneal epithelial and endothelial cells, lens epithelial cells, ciliary epithelial cells, retinal ganglion cells, and the RPE cells. During the development of choroidal neovascularization, PEDF mRNA, PEDF protein, and VEGF protein were strongly detected in many cells within the laser lesions at 3 days after photocoagulation, after which levels gradually declined. However, PEDF was still expressed in the RPE cells that proliferated and covered the neovascular tissues at 2 weeks, whereas VEGF protein was weakly expressed in endothelial cells in choroidal neovascularization. CONCLUSIONS: PEDF is expressed in different cell types of normal rat eyes. The expression of PEDF was detected in the choroidal neovascular tissues induced by photocoagulation, and these findings suggest that PEDF may modulate the process of choroidal neovascularization.     

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.     

Expression of Pigment Epithelium-Derived Factor and Vascular Endothelial Growth Factor in Fibrovascular Membranes from Patients with Proliferative Diabetic Retinopathy

Purpose

The expression of pigment epithelium-derived factor (PEDF), a strong inhibitor of angiogenesis, has not been examined in human ocular fibrovascular membranes, to the best of our knowledge. The purpose of this study was to determine whether PEDF is expressed in the fibrovascular membranes in eyes of patients with proliferative diabetic retinopathy (PDR), and to compare the expression of PEDF with that of vascular endothelial growth factor (VEGF).

Methods

The expression of PEDF and VEGF in the fibrovascular membranes excised during vitreous surgery in eight cases of PDR was determined by immunohistochemistry.

Results

VEGF was strongly expressed in the endothelial cells of newly formed vessels in the fibrovascular membranes. In contrast, PEDF was weakly expressed in the endothelial cells and was prominently expressed in the extracellular matrix and fibrous tissue surrounding the new vessels.

Conclusions

Our results suggest that PEDF, along with VEGF, may modulate the formation of fibrovascular membranes in patients with PDR.?Jpn J Ophthalmol 2006;50:116–120 © Japanese Ophthalmological Society 2006     

The potential angiogenic role of macrophages in the formation of choroidal neovascular membranes.

PURPOSE: To investigate the distribution of inflammatory mediators such as interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha and angiogenic cytokines such as vascular endothelial growth factor (VEGF) and to identify their cellular source in surgically excised choroidal neovascular membranes (CNVMs) of various origins. METHODS: Immunoperoxidase staining was performed on paraffin-embedded sections of 11 surgically excised CNVMs to identify cellular distribution and localization of cytokines. Immunofluorescent double staining was performed to detect the cellular source of cytokines. RESULTS: Cytokeratin-positive cells were detected in the RPE layer, in stromal cells, and around neovascular vessels. Macrophages identified by their cellular marker CD68 showed almost the same distribution as cytokeratin-positive cells, although they were most prominent in the stroma. A substantial number of neovascular vessels were also immunoreactive to IL-1beta and TNF-alpha. Immunofluorescent double staining revealed that the RPE layers immunopositive for cytokeratin were also immunopositive for all cytokines, whereas stromal cells immunostained for CD68 were positive for IL-1beta and TNF-alpha, but not for VEGF. CONCLUSIONS: These results indicate that IL-1beta and TNF-alpha secreted by macrophages may promote, at least in part, angiogenesis in CNVMs by stimulating VEGF production in RPE cells.     

Expressions of angiopoietins and Tie2 in human choroidal neovascular membranes.

PURPOSE: To elucidate the potential role of angiopoietins and the Tie2 system in choroidal neovascularization. METHODS: Surgically excised choroidal neovascular membranes (CNVMs) were obtained at vitrectomy from five eyes with age-related macular degeneration, three eyes with idiopathic neovascular maculopathy, and two eyes had degenerative myopia and two eyes had angioid streaks. Light microscopic immunohistochemistry was performed to detect cytokines such as vascular endothelial growth factor (VEGF), Ang1, and Ang2 and cellular components such as retinal pigment epithelial (RPE) cells, macrophages, and endothelial cells. Immunofluorescent double staining using confocal microscopy was performed to identify the cell types that secrete specific cytokines. RESULTS: Ang1 and Ang2 were positive in all surgically excised CNVMs, regardless of the primary disease. Double staining revealed that many of the cytokeratin, CD68 and factor VIII positive cells also had Ang1 and Ang2 immunoreactivities. In contrast to Ang1, Ang2 immunoreactivity tends to be higher in the highly vascularized regions of many CNVMs, and the localization was very similar to that of VEGF staining. Almost all vascular structures had prominent immunoreactivity for Tie2, which was confirmed by double staining for Tie2 and factor VIII. Tie2 immunoreactivity was also observed in the RPE monolayer and in pigmented, polygonal, and fibroblast-like cells in the stroma. CONCLUSIONS: Present findings that Ang2 and VEGF are co-upregulated and that Tie2 is expressed in a variety of cell types in CNVMs further support a crucial role of the interaction between VEGF and Ang2 in pathologic angiogenesis of CNVM formation.     

Angiogenesis and retinal diseases

Angiogenesis is the process involving the growth of new blood vessels from preexisting vessels which occurs in both physiologic and pathological settings. It is a complex process controlled by a large number of modulating factors, the pro-and antiangiogenic factors. The underlying cause of vision loss in proliferative retinal diseases, such as age-related macular degeneration and proliferative diabetic retinopathy, are increased vascular permeability and choroidal neovascularization, and vascular endothelial growth factor (VEGF) plays a central role in this process. VEGF is produced in the eye by retinal pigment epithelium (RPE) cells and is upregulated by hypoxia. There are four major biologically active human isoforms, of which VEGF165 is the predominant in the human eye and appears to be the responsible for pathological ocular neovascularization. Besides being a potent and specific mitogen for endothelial cells, VEGF increases vascular permeability, inhibits endothelial cells apoptosis, and is a chemoattractant for endothelial cell precursors. VEGF is not the only growth factor involved in ocular neovascularization. Basic fibroblast growth factor (bFGF), angiopoietins, pigment epithelium-derived factor (PEDF), and adhesion molecules also play a role in the pro- and antiangiogenic balance. Advances in the understanding of the bases of pathological ocular angiogenesis and identification of angiogenesis regulators have enabled the development of novel therapeutic agents. Anti-VEGF antibodies have been developed for intravitreal use, and other approaches are currently under investigation. These new drugs may be powerful tools for the treatment of the leading causes of irreversible blindness in people over age 65.     

Neuropilin-1 expression by endothelial cells and retinal pigment epithelial cells in choroidal neovascular membranes

PURPOSE: To determine if vascular endothelial growth factor (VEGF) coreceptor neuropilin-1 (NP-1) is expressed in choroidal neovascularization (CNV) and to localize the expression. DESIGN: Laboratory investigation. METHODS: Six CNV membranes (CNVMs) obtained from patients with subfoveal CNV attributable to age-related macular degeneration (AMD) underwent immunohistochemistry for VEGF receptor-2 (VEGFR-2) and NP-1. The positive cell types were identified by double staining with anticytokeratin, anti-CD31, and antismooth muscle actin (SMA). RESULTS: Immunohistochemical staining revealed positivity for VEGFR-2 and NP-1 in all six CNVMs. Both receptors were strongly expressed by new choroidal endothelial cell forming vessels and CD-31-positive cells in the nonvascular area. They were also expressed in the pigmented retinal pigment epithelial layer and by cytokeratin-positive, nonpigmented retinal pigment epithelium cells in the nonvascular area. The nonpigmented retinal pigment epithelium cells positive for VEGFR-2 and NP-1 were highly colocalized with SMA. CONCLUSIONS: The presence of both VEGF and NP-1 suggest that NP-1 may play a role in the evolution of CNV in AMD.     

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.     

Molecular mechanism of choroidal neovascularization in age-related macular degeneration

The exudative form of age-related macular degeneration (AMD) is characterized by choroidal neovascularization (CNV). Retinal pigment epithelial cells (RPE) secrete various angiogenesis-related factors, especially vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). The imbalance between the VEGF and PEDF secreted by RPE is a key contributor to the development of CNV in AMD. The earliest clinical hallmark of AMD is the presence of drusen. Although drusen are an epidemiological risk factor for the development of CNV, the mechanism of how drusen induce the development of CNV remains unclear. Recent proteome analysis demonstrated that amyloid beta (Abeta) deposition was specific to drusen from eyes with AMD. We focused on Abeta and investigated the effect of Abeta on cultured human RPE cells as well as ocular findings in neprilysin gene-disrupted mice, which leads to an increased deposition of Abeta. Our study demonstrates that Abeta accumulation affects the balance between VEGF and PEDF in the RPE, and reproduces features characteristic of human AMD, such as RPE atrophy and basal deposit formation in neprilysin gene-disrupted mice.     

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.     

Expression of leukocyte adhesion molecules in human subfoveal choroidal neovascular membranes treated with and without photodynamic therapy

PURPOSE: The purposes of this study were to investigate the immunostaining of the leukocyte adhesion molecules intercellular adhesion molecule (ICAM)-1 and E-selectin in subfoveal choroidal neovascular membranes (CNVMs) surgically excised from patients with age-related macular degeneration (AMD) and to determine whether prior photodynamic therapy (PDT) alters their immunostaining. METHODS: The localization of ICAM-1 and E-selectin in 10 subfoveal CNVMs was determined by immunohistochemistry. Membranes were also immunostained for CD31 to assess vascularity. RESULTS: Significantly higher numbers of CD31-staining vessels per unit membrane area were found in the peripheral regions of the membranes compared with the central regions (P = 0.05). ICAM-1 immunoreactivity in the CNVMs was found predominantly on RPE cells, but also on small vessels in the periphery. ICAM-1 staining was significantly more intense in the peripheral, more cellular areas of the membranes than in the central, more fibrotic regions (P = 0.04). ICAM-1 staining in the periphery of the CNVMs was greater than that in choroidal vessels and the RPE of the normal control eye. ICAM-1 immunostaining grade in peripheral regions of the CNVMs decreased with the increasing number of PDT treatments (P = 0.05). Some of the CNVMs also stained for E-selectin in RPE cells and small vessels in the periphery. CONCLUSIONS: In subfoveal CNVMs from patients with AMD, there is increased immunostaining for leukocyte adhesion molecules, particularly in the peripheral, more cellular regions where angiogenesis may be ongoing. Increasing numbers of PDT treatments may be associated with decreased ICAM-1 immunostaining in the proliferating edges of the CNVMs.     

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)     

脉络膜黑色素瘤中血管内皮生长因子与色素上皮衍生因子的关系及意义

目的 观察脉络膜黑色素瘤中血管内皮生长因子（VEGF）和色素上皮衍生因子（PEDF）的表达,探讨两者在脉络膜黑色素瘤血管生成、肿瘤生长和浸润转移中的作用。方法 用免疫组织化学SP法检测58例脉络膜黑色素瘤石蜡组织标本中VEGF和PEDF蛋白的表达,用原位杂交法检测其中39例石蜡组织中PEDF mRNA的表达。结果 58例脉络膜黑色素瘤石蜡组织中PEDF蛋白阳性表达率为22.4%,VEGF阳性表达率为74.1%;PEDF蛋白在巩膜导管未受侵犯组阳性率高于巩膜导管受侵犯组,差异有统计学意义（P＜0.05）;VEGF蛋白在巩膜导管未受侵犯组阳性表达率低于巩膜导管受侵犯组,差异有统计学意义（P＜0.05）。39例脉络膜黑色素瘤石蜡组织中PEDF mRNA阳性表达率为46.2%,巩膜导管未受侵犯组PEDF mRNA阳性率高于巩膜导管受侵犯组,其差异有统计学意义（P＜0.05）。结论 VEGF和PEDF的失衡在脉络膜黑色素瘤血管发生、肿瘤的生长和浸润转移中起着重要的作用。     

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 neuropilin-1 in choroidal neovascular membranes

BACKGROUND: Neovascularization is a serious consequence of several eye diseases, including age-related macular degeneration. Neovascularization is under the control of proangiogenic factors, such as vascular endothelial growth factor and fibroblast growth factor. Recent work in our laboratory has focused on other, novel angiogenic factors, such as neuropilin-1, and their potential role in neovascularization. The purpose of this study was to investigate the role of neuropilin-1 in choroidal neovascularization (CNV). METHODS: We examined the localization of neuropilin-1 by immunohistochemistry in nine choroidal neovascular membranes (CNVMs) surgically excised from four patients with age-related macular degeneration who had not undergone laser photocoagulation, four with idiopathic CNV and one with ocular histoplasmosis. We also stained the membranes for markers of endothelial and retinal pigment epithelial cells. Controls included omission of primary antibody, use of an irrelevant primary antibody, and neuropilin-1 staining of the posterior sclera, choroid and retina of four healthy donor eyes. RESULTS: Neuropilin-1 was present in eight of the nine CNVMs. It was localized mainly to the plasma membrane. The more vascular membranes and those consisting of a larger number of retinal pigment epithelial cells were associated with greater neuropilin-1 staining. Neuropilin-1 was not seen in the posterior segment of the four healthy eyes. INTERPRETATION: Neuropilin-1 appears to play an active role in CNV. Further study is needed to establish a causal relation.     

Expression of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in sickle cell retina and choroid

Pigment epithelium-derived factor (PEDF) has been shown to be an inhibitor of angiogenesis as well as a multipotent neurotrophic factor in the mammalian eye. Changes in PEDF levels have been correlated with development of retinal neovascularization in oxygen-induced retinopathy. The purpose of this study was to determine the localization and relative level of PEDF in human retinas and choroids using immunohistochemistry and evaluate the changes in PEDF and vascular endothelial growth factor (VEGF) localization and their relation to the progression of proliferative sickle cell retinopathy.Cryopreserved tissues from eyes of normal subjects and subjects with non-proliferative or proliferative sickle cell retinopathy were used with streptavidin peroxidase immunohistochemistry. A rabbit polyclonal antibody was made against recombinant human PEDF. Binding of the antibody was blocked by preincubation of the antibody with excess human recombinant PEDF. Relative levels of immunoreactivity were scored with a seven-point grading system and by microdensitometric analysis.The most prominent sites of PEDF localization in the normal eye were the vitreous condensed at the internal limiting membrane and RPE-Bruch's membrane-choriocapillaris complex. PEDF was also prominent in choroidal stroma. There was limited immunoreactivity in some cells of the neural retinas, in blood vessels and in the interphotoreceptor matrix (IPM). There was no difference in ratio (1.47 vs. 1.44) of PEDF/VEGF or the relative levels of either growth factor in the retinal vasculatures of the control subjects and perfused area of non-proliferative sickle cell retinas. The ratio was increased in the non-perfused area of the non-proliferative sickle cell retinas (2.24). In eyes with proliferative sickle cell retinopathy, elevated PEDF and VEGF immunostaining was present in viable vessels of sea fan neovascular formations as well as feeder vessels of sea fans. The PEDF/VEGF ratio in sea fans was 1.0. Immunoreactivity for PEDF was prominent in retinal vessels in non-perfused regions and in atrophic sea fans, while VEGF immunoreactivity was weak or absent in these structures.In conclusion, PEDF and VEGF were both significantly elevated in viable sea fan formations in sickle cell disease (p<0.05) but only PEDF was present in non-viable sea fans. The highest levels of PEDF in all eyes were associated with extracellular matrices (vitreous, choroidal stroma, IPM, and walls of blood vessels). PEDF might play an important role in inhibiting angiogenesis and inducing the regression of sea fans. Progression of angiogenesis may be dependent on the ratio of PEDF/VEGF.     

Peroxisome proliferator-activated receptor-gamma ligands inhibit choroidal neovascularization

PURPOSE: To determine the antiangiogenic effects of peroxisome proliferator-activated receptor (PPAR)-gamma agonists on ocular cells involved in the pathogenesis of choroidal neovascularization (CNV) in vitro and on experimental laser photocoagulation-induced CNV in vivo. METHODS: PPAR-gamma expression in human retinal pigment epithelial (RPE) cells and bovine choroidal endothelial cells (CECs) was determined using an RNase protection assay and Western blot analysis. Two PPAR-gamma ligands, troglitazone (TRO) and rosiglitazone (RSG; 0.1-20 microM), were used to assess effects on RPE and CEC proliferation and migration and CEC tube formation in response to vascular endothelial growth factor (VEGF). The effects of intravitreal injection of TRO on laser photocoagulation-induced CNV lesions in rat eyes (15 experimental, 15 control, nine burns per eye) and cynomolgus monkey eyes (two experimental, two control, seven paramacular burns per eye) was assessed by fluorescein angiography and histologic evaluation. RESULTS. PPAR-gamma1 was expressed in both RPE and CEC. PPAR-gamma ligands significantly inhibited VEGF-induced migration and proliferation in both cell types and tube formation of CEC in a dose-response manner. CNV in rats was markedly inhibited by intravitreous injection of TRO (P < 0.001). Lesions showed significantly less fluorescein leakage and were histologically thinner in the TRO-treated animals. Similar findings were present in the TRO-treated lesions in two monkey eyes. The drug showed no apparent adverse effects in the adjacent retina or in control eyes. CONCLUSIONS: The inhibition of VEGF-induced choroidal angiogenesis in vitro, and CNV in vivo by PPAR-gamma ligands suggests the potential application of these agents in the large group of patients with age-related macular degeneration complicated by CNV.     

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.