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.     

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 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.     

Colocalization of Tie2, angiopoietin 2 and vascular endothelial growth factor in fibrovascular membrane from patients with retinopathy of prematurity

PURPOSE: The tyrosine kinase receptor Tie2 and its ligands, the angiopoietins (Angs), play important roles in vascular integrity and neovascularization, modulating vascular endothelial growth factor (VEGF) activity. To elucidate the potential role of Angs and the Tie2 system in retinopathy of prematurity (ROP), we have investigated the expression of Angs, Tie2 and VEGF within fibroproliferative membranes in ROP. METHODS: Fibroproliferative membranes were obtained from 38 cases with stage 5 ROP at the time of vitrectomy. Membranes were fixed in formalin and embedded in paraffin. Each specimen was serially sectioned for immunohistochemistry. Polyclonal antibodies specific for Ang1, Ang2, Tie2 and VEGF were used for immunostaining. Immunoreactivity for von Willebrand factor (factor VIII) was also assessed to confirm the identity of vascular endothelial cells. RESULTS: Positive staining for Tie2 was observed in 23 of 38 specimens (60.5%). Tie2 was localized in vascularized regions of fibrovascular membranes and was co- expressed with VEGF and factor VIII. Ang2 stained positively in 18 of 38 (47.3%) serial sections where Tie2 was present, and was also co-expressed with VEGF and factor VIII. Ang1 was not generally observed in these specimens (3/38). CONCLUSIONS: VEGF and Ang2-Tie2 interactions may play an important role in the pathogenesis of ROP.     

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.     

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.     

Expression of extradomain-B-containing fibronectin in subretinal choroidal neovascular membranes

PURPOSE: To investigate the presence of the fibronectin isoform containing the extradomain B (B-FN), a marker-protein of angiogenesis, in surgically excised human choroidal neovascular membranes (CNVM) to evaluate whether B-FN could be used as a therapeutic target for specific antibody-photosensitizer immunoconjugates. DESIGN: Laboratory investigation. METHODS: The setting was an institutional practice. The study population consisted of 15 eyes (15 patients) with CNVM undergoing membrane excision (four eyes with age-related macular degeneration, seven with pathologic myopia and four with multifocal choroiditis). The control group consisted of eight eye bank eyes (four subjects) without choroidal neovascularization. Light microscopic immunohistochemistry on cryostat sections of tissues was obtained. B-FN was detected by a human recombinant antibody, CGS-1, and compared with immunostaining for endothelial cells with factor VIII-related antigen. The main outcome measure was the presence of CGS-1 positively stained cells or areas of the extracellular matrix. Staining of CGS-1 was scored on a scale from 0 to 3. RESULTS: Fourteen of 15 neovascular membranes stained strongly with CGS-1 (score 2 or 3). One membrane from a patient with pathologic myopia was negatively stained (score 0). CGS-1 positive staining was detected around endothelial cells and in the extracellular matrix of CNVMs. The retina of eyes without choroidal neovascularization was negative with CGS-1 in all eight donor eyes, while the choroid contained some weakly CGS-1 positive cells (score 0 and 1, respectively). CONCLUSIONS: The extradomain B is abundantly expressed in CNVMs, but its expression is more restricted in eyes harboring no apparent choroidal neovascularization. In the future, B-FN might serve as a target for the delivery of antibody-photosensitizer immunoconjugates to newly developed vessels to enhance the selectivity of photodynamic therapy.     

Expression of estrogen receptor in the choroidal neovascular membranes in highly myopic eyes

PURPOSE: To investigate the expression of estrogen receptor in choroidal neovascular membranes (CNVMs) surgically excised from eyes with high myopia. METHODS: The CNVMs were surgically excised from two eyes with high myopia. Immunohistochemical analysis with a monoclonal antibody to estrogen receptor and in situ hybridization with an oligodeoxynucleotide sequence coding for estrogen receptor were used to study the cellular distribution of estrogen receptor and its messenger RNA in the CNVMs. Immunohistochemical localization of glial fibrillary acidic protein and vimentin in the CNVMs was compared with localization of estrogen receptor. RESULTS: Immunohistochemical analysis with monoclonal antibody to estrogen receptor showed widespread staining throughout the CNVMs. By in situ hybridization, the expression of estrogen receptor messenger RNA was predominantly observed in the CNVMs. Staining with antibody to vimentin was widespread throughout the CNVMs, which was similar to the localization of estrogen receptor. CONCLUSION: Estrogen receptor was expressed in the CNVMs in highly myopic eyes, suggesting that estrogen may have important functions in the formation of CNVMs.     

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.     

Thrombospondin-1 Expression in RPE and Choroidal Neovascular Membranes

Introduction Age-related macular degeneration (AMD) s the leading cause of blindness in the West or individuals more than 50 years of age[1-3].Severe visual loss in the late stages of AMD most commonly results from choroidal neovas- cularization (CNV), a process characterized by the growth of new vessels from the choriocapil- laris through Bruch′s membrane. These new vessels are prone to leakage and bleeding and may be associated with detachment of the retinal pigment epithelium (RPE). …     

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.     

Correlation of Indocyanine Green Angiography Findings and Expression of Vascular Endothelial Growth Factor in Surgically Excised Age-related Macular Degeneration-related Choroidal Neovascular Membranes

Purpose: We investigated the relationship between clinical classification by indocyanine green angiography (IA) and pathologic findings including the expression of vascular endothelial growth factor (VEGF) in age-related macular degeneration-related choroidal neovascular membranes.Subject and Methods: The subjects were 15 patients with age-related macular degeneration who underwent surgical excision for choroidal neovascular membrane. The patients were classified into 4 types: Type I, hyperfluorescence in both early and late phases (n = 7); Type II, hyperfluorescence in the early phase only (n = 2); Type III, hyperfluorescence in the late phase only (n = 3); and Type IV, no hyperfluorescence in any phase (n = 3). The excised choroidal neovascular membranes were fixed and stained by hematoxylin-eosin and azan. They were also examined by immunohistochemical staining for VEGF.Results: VEGF was expressed markedly in vascular endothelial cells and fibroblast-like cells of interstitial tissue of Types I, II and III. Its expression was weak in Type IV.Conclusion: Clinical classification by IA for age-related macular degeneration is consistent with the pathologic findings including the expression of VEGF.     

Correlation of indocyanine green angiography findings and expression of vascular endothelial growth factor in surgically excised age-related macular degeneration-related choroidal neovascular membranes

PURPOSE: We investigated the relationship between clinical classification by indocyanine green angiography (IA) and pathologic findings including the expression of vascular endothelial growth factor (VEGF) in age-related macular degeneration-related choroidal neovascular membranes. SUBJECT AND METHODS: The subjects were 15 patients with age-related macular degeneration who underwent surgical excision for choroidal neovascular membrane. The patients were classified into 4 types: Type I, hyperfluorescence in both early and late phases (n = 7); Type II, hyperfluorescence in the early phase only (n = 2); Type III, hyperfluorescence in the late phase only (n = 3); and Type IV, no hyperfluorescence in any phase (n = 3). The excised choroidal neovascular membranes were fixed and stained by hematoxylin-eosin and azan. They were also examined by immunohistochemical staining for VEGF. RESULTS: VEGF was expressed markedly in vascular endothelial cells and fibroblast-like cells of interstitial tissue of Types I, II and III. Its expression was weak in Type IV. CONCLUSION: Clinical classification by IA for age-related macular degeneration is consistent with the pathologic findings including the expression of VEGF.     

Expression of angiogenic factors by photodynamic therapy

PURPOSE: The purpose of this study was to evaluate the impact of photodynamic therapy (PDT) on the regulation of angiogenic factors such as vascular endothelial growth factor (VEGF) and pigment epithelium derived factor (PEDF) in human eyes. METHODS: Eyes of patients with untreatable malignancy served as the study eyes (n = 2), age-matched donor eyes were used as controls (n = 2). Standard Verteporfin-PDT was applied to intact areas of the posterior pole. One week after PDT the eyes were examined by ophthalmoscopy as well as fluorescein (FA) and indocyanine green angiography (ICGA). After enucleation the eyes were processed for LM/EM histology. Immunolabeling using specific antibodies against VEGF and PEDF was performed in PDT-treated areas, untreated collateral areas of study eyes and untreated areas of control eyes. RESULTS: All PDT-treated areas demonstrated a typical choroidal hypofluorescence on FA/ICGA. In LM/EM histology a selective damage of choriocapillary endothelial cells was found. VEGF expression was localized only to choriocapillary endothelial cells and focally in larger choroidal vessels of PDT-treated areas. Untreated areas of study eyes and controls were VEGF-negative. PEDF staining was observed in retinas and RPE of all eyes, but only choroidal endothelial cells of PDT-treated areas showed a PEDF-positive reactivity. CONCLUSION: PDT not only induces structural and angiographic, but also biological effects in human eyes. VEGF and PEDF expression can be documented in choroidal endothelial cells following PDT and could have an impact on the recovery process after treatment.     

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.     

Regression of ocular neovascularization in response to increased expression of pigment epithelium-derived factor

PURPOSE: Several pharmacologic treatments have been shown to reduce ocular neovascularization when administered before the onset of angiogenic stimuli, but none have been shown to cause regression of already established ocular neovascularization. In this study, the authors tested the effect of adenoviral vectored pigment epithelium-derived factor (PEDF) gene transfer on established neovascularization in transgenic mice with expression of vascular endothelial growth factor (VEGF) in photoreceptors (rho/VEGF mice) and in a model of choroidal neovascularization. METHODS: Two weeks after the onset of VEGF transgene expression in rho/VEGF mice or 2 weeks after laser-induced rupture of Bruch's membrane in wild-type mice, subgroups of mice were killed, and the baseline amount of neovascularization was measured by image analysis. The remainder of the mice received an intravitreous or subretinal injection of adenoviral vector containing a PEDF expression construct (AdPEDF.11) or control vector (AdNull.11). RESULTS: Seven days after injection in rho/VEGF mice or 10 days after injection in the choroidal neovascularization model, the amount of neovascularization in AdPEDF.11-injected eyes was significantly less than the baseline level, indicating that regression of neovascularization had occurred. There was TUNEL staining within choroidal neovascular lesions in eyes injected with AdPEDF.11. Eyes given a subretinal injection of AdNull.11 had TUNEL-positive cells in the retina, but none within areas of choroidal neovascularization. CONCLUSIONS: These data indicate that increased expression of PEDF causes regression of ocular neovascularization by promoting apoptosis of cells within neovascular lesions and possibly represents a new treatment paradigm for patients with established ocular neovascularization.     

Insulin-like growth factor-I and its receptor in neovascular age-related macular degeneration

PURPOSE: The insulin-like growth factor (IGF)-I protein is a growth-promoting polypeptide that can act as an angiogenic agent in the eye. The purpose of the current study was to localize the expression of IGF-I and its receptor (IGF-IR) mRNA and IGF-IR protein in situ in the normal human eye and to examine the presence of expression in eyes with neovascular age-related macular degeneration (AMD). METHODS: Formalin-fixed, paraffin-embedded slides of 4 normal control eyes and 14 eyes with choroidal neovascularization (CNV) secondary to AMD were examined. Three eyes with proliferative diabetic retinopathy were studied as the positive control. IGF-I and IGF-IR mRNA was detected by in situ hybridization with digoxigenin-labeled RNA probes. IGF-IR protein was studied by immunohistochemistry. RESULTS: In the normal retina, IGF-I and IGF-IR mRNA expression was found throughout the neuroretinal layers, in the retinal pigment epithelium (RPE), and in some choriocapillary and retinal capillary endothelial cells. In eyes with CNV we found IGF and IGF-IR mRNA in capillary endothelial cells, some transdifferentiated RPE, and fibroblast-like cells. IGF-IR protein was found in normal eyes in all neuroretinal layers, in the RPE, and in the choroidal vessels. In eyes with CNV, IGF-IR protein was present in the RPE monolayer, in transdifferentiated RPE, and in newly formed vessels. CONCLUSIONS: The colocalization of protein and receptor indicates an autocrine function of IGF-I in the normal human retina. Because IGF-I participates in ocular neovascularization, synthesis of IGF-IR and IGF-I in endothelial cells, RPE cells, and fibroblast-like cells in CNV may point toward a role for this growth factor in the pathogenesis of neovascular AMD.     

Comparison with indocyanine green angiography and immunohistochemical study of choroidal neovascular membrane in age-related macular degeneration

PURPOSE: We classified choroidal neovascular membranes (CNMs) (48 eyes) in age-related macular degeneration (AMD) into four types using indocyanine green angiography. METHODS: Surgically extracted CNMs were examined immunohistochemically. Specimens were stained with glial fibrillary acidic protein (GFAP), von Willebrand factor, Ki-67 antigen, actin, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (b-FGF), and transforming growth factor (TGF-beta 1). RESULTS: All types of CNM were positively stained with GFAP on the side opposite the retinal pigment epithelial (RPE) cells. This suggests the adherence of neurosensory retina to the CNM. Type I membranes which showed hyperfluorescence in the early and late phase of ICG angiography were significantly stained with Ki-67 antigen. CONCLUSION: These results indicate that CNMs in AMD are extracted with neurosensory retina and RPE cells during surgery. Type I membranes have a high capability for proliferation.