May 2003
Volume 44, Issue 5
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Retina  |   May 2003
Insulin-like Growth Factor-I and its Receptor in Neovascular Age-Related Macular Degeneration
Author Affiliations
  • Antoinette C. Lambooij
    From the Departments of Ophthalmology,
  • Karel H. M. van Wely
    Experimental Pathology,
  • Dicky J. Lindenbergh-Kortleve
    Pediatrics, and
  • Robert W. A. M. Kuijpers
    From the Departments of Ophthalmology,
  • Mike Kliffen
    Pathology, Erasmus Medical Center Rotterdam, The Netherlands; and the
  • Cornelia M. Mooy
    Pathology, Erasmus Medical Center Rotterdam, The Netherlands; and the
    Pathology Laboratory, Dordrecht, The Netherlands.
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 2192-2198. doi:10.1167/iovs.02-0410
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      Antoinette C. Lambooij, Karel H. M. van Wely, Dicky J. Lindenbergh-Kortleve, Robert W. A. M. Kuijpers, Mike Kliffen, Cornelia M. Mooy; Insulin-like Growth Factor-I and its Receptor in Neovascular Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2003;44(5):2192-2198. doi: 10.1167/iovs.02-0410.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

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.

Neovascular age-related macular degeneration (AMD) is characterized by choroidal neovascularization (CNV), in which newly formed vessels from the underlying choroid grow beneath the retinal pigment epithelium (RPE) and the neuroretina. CNV may cause (sub) acute blindness because of bleeding or scar formation. 1 Although the morphology of angiogenesis in CNV secondary to AMD has been described in detail, the pathogenesis is still poorly understood. 
Growth factors are acknowledged to play an important role in retinal neovascularizations. Vascular endothelial growth factor (VEGF), an endothelium-specific mitogen, is regarded as one of the most important ocular angiogenic factors, especially under hypoxic circumstances. 2 3 Other angiogenic factors in ocular neovascularization include basic fibroblast growth factor, transforming growth factor-β, platelet-derived growth factor, and insulin-like growth factor (IGF)-I. 2 4  
In an earlier study, we demonstrated that most CNVs in AMD express somatostatin receptor type 2A, which binds potential antiangiogenic somatostatin analogues. 5 Somatostatin receptors are present on cultured human retinal endothelial cells. Proliferation of retinal endothelial cells 6 and choroidal endothelial cells 7 can be inhibited by somatostatin analogues. In mice, inhibition of IGF-I by somatostatin analogues or by downregulation of growth hormone (GH) can decrease ischemia-induced retinal neovascularization. 8 IGF-I is a growth-promoting polypeptide that has mitogenic and differentiating effects on many cell types, among which are ocular vascular endothelial cells, 9 RPE cells, 10 11 and neuronal cells. 12 IGF-I can act as a direct angiogenic factor on retinal endothelial cells, 6 7 or indirectly through increased VEGF gene expression of cultured RPE cells. 13  
Most studies describe the role of IGF-I in in vitro models 6 7 14 15 16 17 or in situ in diabetic retinopathy. 18 19 So far, IGF-I has not been studied in ARM. 
The purpose of our study was to localize the expression of IGF-I and its receptor (IGF-IR) mRNA and IGF-IR protein in situ in normal human eyes and to examine the presence of expression in eyes with CNV, to elucidate its possible role in angiogenesis in AMD. 
Materials and Methods
The study was performed according to the tenets of the Declaration of Helsinki. Enucleation or surgical excision of subfoveal CNV was performed after obtaining the informed consent of the patient. 
Patients
All eyes were retrieved from the Ophthalmic Pathology Department of the University Hospital of Rotterdam. Four enucleated eyes without ischemic disease (enucleated for other reasons) and three enucleated eyes with proliferative diabetic retinopathy (PDR) were used as the control. Fourteen eyes (five enucleated eyes, four donor eyes and five surgically removed subretinal neovascular membranes) of 13 patients with neovascular AMD were studied. The clinical and histologic diagnosis of the eyes and the classification of CNV are described in Table 1 . The eyes were processed for routine diagnostic procedures by fixation in 10% buffered formaldehyde and were embedded in paraffin. Five-micrometer sections were prepared for in situ hybridization and immunohistochemistry. 
RNA Probes
The human IGF-I probe was a 258-bp fragment containing exon 2 and 3 of the IGF-I gene. 20 The human IGF-IR probe was generated using a cDNA clone with a unique insert of human IGF-IR (IMAGE cDNA clone 150361, Research Genetics, Huntsville, AL). The insert was reduced to 270 bp by restriction with BamHI and AvaI and ligated in antisense and sense vectors (pBluescript SK and KS, respectively; Stratagene Europe, Amsterdam, The Netherlands). Escherichia coli X12Blue was transformed with these vectors, and proper colonies were isolated and grown. Sequence analysis was performed to verify the inserts. Digoxigenin-11-UTP labeled RNA probes were prepared according to the manufacturer’s instructions (Roche Diagnostics, Mannheim, Germany), using T7 RNA polymerase. 
In Situ Hybridization
Sections were deparaffinated with xylene and rehydrated. The slides were incubated in the following solutions: 0.2 N HCl, 0.3% Triton X-100 in phosphate-buffered saline (PBS), RNase-free proteinase K (5 μL/mL for 20 minutes at 37°C) and 4% formalin in PBS. Subsequently, acetylation was performed with acetic anhydride in 0.1 M triethanolamine. The slides were rinsed in 2× SSC (1× SSC containing 150 mM NaCl and 15 mM sodium citrate) and preincubated in 50% formamide in 2× SSC at 37°C. For hybridization, antisense and sense probes were diluted in hybridization solution (50% deionized formamide, 10% dextran sulfate, 4× SSC [IGF-I] or 2× SSC [IGF-IR], 1× Denhardt’s solution, 1 μg/mL tRNA, and 250 μg/mL herring sperm RNA) to a concentration of 400 ng/mL, and incubated at 68°C for 30 minutes. The hybridization solution was then layered onto the sections and hybridized overnight at 55°C in a humid chamber. Posthybridization washes were performed at 45°C for 30 minutes in the following solutions: 50% formamide in 2× SSC, 50% formamide in 1× SSC, 0.1× SSC (IGF-IR), or 0.5× SSC (IGF-I). The slides were incubated with RNase T1 (2 U/mL) in 2× SSC and 1 mM EDTA in 37°C for 15 minutes and washed at 45°C with 1× SSC and at room temperature with 2× SSC. The digoxigenin-labeled hybrids were detected by antibody incubation performed according to the manufacturer’s instructions (Roche Diagnostics) with the following modifications. A 1:1000 dilution of anti-digoxigenin (Fab) conjugated to alkaline phosphatase was used for a 2.5-hour incubation at room temperature or overnight at 4°C. Afterward, an extra washing step of 0.025% Tween in Tris-buffered saline (pH 7.5) was performed. For staining, sections were layered with detection buffer (0.1 M Tris-HCl, 0.1 M NaCl, 0.05 M MgCl2 [pH 9.5]) containing 4-nitroblue tetrazolium chloride (NBT), 5-bromo-4-chloro-3-indolyl-phosphate (BCIP; both from Vector, Burlingame, CA) and 6% polyvinylalcohol (molecular weight 31,000 to 50,000, from Aldrich Chemical, Milwaukee, WI). The color reaction was performed in the dark and was stopped when the desired intensity of the resultant blue precipitate was reached. Sections were washed in 10 mM Tris-HCl and 1 mM EDTA (pH 8.0), counterstained with nuclear red solution, dehydrated with ethanol gradients, and mounted. 
Immunohistochemistry
Polyclonal rabbit antibodies against IGF-IR (1:750 dilution) were obtained from Research Diagnostics (Flanders, NJ). Immunohistochemistry against IGF-I was not performed, because adequate antibodies for paraffin-embedded material are not available. Antibodies against pankeratin (monoclonal mouse antibodies; 1:100 dilution); factor VIII (monoclonal mouse antibodies; 1:50 dilution), and glial fibrillary acidic protein (GFAP; polyclonal rabbit antibodies; dilution 1:200) were obtained from Dako (Glostrup, Denmark). Monoclonal mouse antibodies against smooth muscle actin (SMA; 1:150 dilution) were obtained from Biogenex (San Ramon, CA). Sections were deparaffinated and rehydrated. Antigen retrieval was performed for the IGF-IR and pankeratin antibodies (microwave heating for 10 minutes) and for the factor VIII antibodies (pronase treatment for 20 minutes at 37°C). After the slides were blocked with normal goat serum (Dako, 1:10) for 15 minutes, they were incubated with the primary antibodies for 1 hour at room temperature or overnight at 4°C (IGF-IR). The sections were further incubated with biotinylated multilink antibodies for 30 minutes, followed by alkaline phosphatase-labeled antibiotin (both Biogenex) for 30 minutes. The complex was visualized by incubation with new fuchsin (as a red chromogen) for 30 minutes in the dark. The slides were counterstained with Mayer hematoxylin, mounted, and examined by light microscopy. Negative controls for immunohistochemistry included (1) omission of the primary antibody, (2) incubation with an irrelevant polyclonal rabbit antibody, and (3) preabsorption of the IGF-IR antibodies with ten times the amount of the immunizing IGF-IR peptide as of the antibody (Research Diagnostics) for 4 hours. 
Grading of Expression and Statistics
The slides were examined by light microscopy. Grading of mRNA and IGF-IR protein expression was performed in a masked fashion by two authors (ACL, CMM) twice and once, respectively. Slides were blinded and randomly graded per cell type in three categories (Table 2) : (1) no staining, (2) staining in less than 50% of cells, (3) staining in more than 50% of cells. 
Positive cell types in CNV were identified with pankeratin staining (RPE cells), factor VIII staining (vascular endothelial cells), GFAP staining (neuronal cells), and SMA (myofibroblasts) in consecutive slides, combined with cellular morphology at examination by light microscopy. Cells that did not meet these criteria were classified as other. 
For inter- and intraobserver variability, a κ statistic was calculated. 
Results
In two eyes with neovascular AMD (Table 2 ; eyes CNV8 and CNV13) the normal retina, regarded as positive internal control, was negative with in situ hybridization. All mRNA appeared to be lost in these eyes. 
In Situ Hybridization
IGF-I.
In the normal retina, IGF-I mRNA expression (Figs. 1A 1D) was found in the ganglion cell layer, inner nuclear layer, outer limiting membrane, RPE monolayer, and some cells in the choroid. Choriocapillary endothelial cells and retinal vessels were positive infrequently. Further expression was found in the lens epithelium, and in all corneal layers (not shown). Hybridization with the sense probe was negative. In eyes with CNV we found IGF mRNA in the retina in the same pattern as in normal retina. Staining in the preexistent RPE monolayer was similar to normal eyes. In 8 of 14 eyes with CNV, expression was found in vascular endothelial cells, some RPE cells, and fibroblast-like cells (Table 2 ; Figs. 2A 2D 2G 2J ). Eyes with PDR showed identical expression in retinal layers. In the preretinal membranes, endothelial cells from newly formed capillaries and fibroblast-like cells stained positive (Fig. 1G) . Hybridization with the sense probe was negative (Fig. 2L)
IGF-I Receptor.
In the normal eye mRNA of the IGF-IR (Figs. 1B 1F) was seen in the ganglion cell layer, inner and outer nuclear layer, and outer limiting membrane. The RPE was strongly positive. Endothelial cells of the choriocapillaris and the choroidal and intraretinal vessels were frequently positive. Further expression was found in the nonpigmented epithelium and to a lesser extent in the pigment epithelium of the ciliary body, the iris dilator muscle, the iris pigment epithelium, and iris endothelial cells. The lens epithelium and all corneal layers were also positive. Hybridization with the sense probe was negative. In all eyes with CNV, we found IGF-IR mRNA in endothelial cells of newly formed vessels, in RPE-cells and in the RPE monolayer, and in fibroblasts (Table 2 ; Figs. 2B 2E 2H 2K ). Eyes with PDR showed expression in retinal layers and RPE similar to normal eyes. In the diabetic preretinal membranes (Fig. 1H) endothelial cells from newly formed capillaries and fibroblast-like cells were positive. Hybridization with the sense probe was negative (Fig. 2M)
Immunohistochemistry
In the normal eyes, we found IGF-IR protein in the choroidal vessels, in the RPE, and in all layers of the neuroretina (Figs. 1C 1F) ; the choriocapillaris was negative. The negative controls showed no staining, except for the peptide control, in which staining of the RPE monolayer was not totally blocked compared with retinal staining, indicating an aspecific component in the RPE staining. In all eyes with CNV, we found the IGF-IR protein in RPE cells, and in 9 of 13 classifiable eyes, staining was present in newly formed vessels (Table 2 ; Figs. 2C 2F 2I ). Eyes with PDR showed expression in the retina similar to that in normal eyes, and, in the diabetic preretinal membranes, endothelial cells from newly formed capillaries were positive (Fig. 1I)
Statistics
Grading of mRNA and IGF-IR protein expression was performed in a masked fashion by two authors (ACL, CMM), twice, with an interval of 7 weeks, and once, respectively. For interobserver and intraobserver variabilities, κ was 0.75 and 0.83, respectively. 
Discussion
In the normal human retina, we found IGF-I and IGF-IR mRNA expression throughout the neuroretina in the same pattern, as described in other species. 21 22 Expression in human RPE has not been described before in situ, but has been detected in cultured human RPE cells. 14 15 16 IGF-I stimulates differentiation and proliferation of vascular endothelial cells, 23 RPE cells, 16 and neural retinal cells 12 24 and is also neuroprotective. 24 25 26 Our finding of protein and receptor colocalization confirms a paracrine–autocrine function of IGF-I in the normal human retina. 21 27  
The observed hybridization signal of IGF-IR was much stronger than that of IGF-I. This may represent a real difference in expression, confirming the findings of higher IGF-IR expression in cultured human RPE. 14 15 However, intensity of staining is not a reliable quantitative criterion, and the IGF-IR probe may be more sensitive than the IGF-I probe. 
In CNV secondary to ARM, we demonstrated mRNA expression of IGF-I and IGF-IR in vascular endothelial cells, in RPE cells, and in fibroblast-like cells. Similarly, we found expression of IGF-I mRNA and its receptor in epiretinal membranes in eyes with PDR, including endothelial cells from newly formed capillaries and in fibroblast-like cells. This is in accordance with a previous diabetic retinopathy study, in which binding sites for IGF-I (receptors or binding proteins) were demonstrated in vessel walls, as well as in cells in fibrous tissue in human diabetic epiretinal membranes. 18 In human and rat diabetic retinas, IGF-I mRNA levels are decreased, 19 28 but vitreous levels of IGF-I are elevated. 29 30 31 32 33 Our findings confirm that intraocular synthesis may contribute to these elevated concentrations. 32  
The pathogenesis of neovascular AMD involves the choroidal vasculature rather than the retinal vasculature, as in diabetic retinopathy. In vitro, IGF-I stimulates the proliferation of choroidal endothelial cells. 7 Therefore, synthesis of IGF-IR and IGF-I in endothelial and RPE cells in CNV may point toward a role for this growth factor in the pathogenesis of neovascular AMD, because both cell types appear to have an important role in this process. 1  
IGF-I participates in ocular neovascularization. 23 It is involved in the degradation of basement membranes, and proteolysis of extracellular matrix, and in migration and proliferation of vascular endothelial cells. 6 Intravitreous injection of IGF-I produces preretinal neovascularization in rabbits 34 or microangiopathy resembling diabetic microangiopathy in pigs. 35 IGF-I also increases RPE cell migration and proliferation in vitro. 11 Furthermore, IGF-I induces upregulation of VEGF mRNA expression in RPE cells 13 and fibroblasts, 36 which in turn also stimulates endothelial cells. 13 Antagonism of IGF-IR suppresses retinal neovascularization and reduces the retinal endothelial cell response to VEGF, 37 which allows for the hypothesis that IGF-I has a permissive role in VEGF-induced neovascularization. 
Inhibition of IGF-I, achieved with somatostatin analogues, 8 may occur at different levels. First, somatostatin inhibits the GH-IGF axis. Furthermore, somatostatin can inactivate the mitogenic potential of IGF-I directly by inactivating the phosphorylated, active form of IGF-IR. 23 38 Finally, somatostatin can act directly as an antiangiogenic agent through binding to somatostatin receptors. 6 Therefore, somatostatin analogues may be an effective therapy for neovascular AMD. 
IGF-I is recruited in normal wound repair. 39 40 This may partly explain the presence of IGF-I in CNV, because formation of the disciform lesion is regarded as normal wound repair. 1  
In conclusion, in this descriptive study we localized the expression of IGF-I mRNA and IGF-IR protein and mRNA in the normal eye. The colocalization of protein and receptor indicates an autocrine function of IGF-I in the human retina. Furthermore, we detected synthesis of both IGF-I and its receptor, and IGF-IR protein in ocular neovascular membranes of patients with AMD and patients with diabetes. Because IGF-I participates in ocular neovascularization, synthesis of IGF-IR and IGF-I in vascular endothelial, RPE, and fibroblast-like cells in CNV may indicate a role for this growth factor in the pathogenesis of neovascular AMD. The exact role of the IGF family in CNV and its possible therapeutic possibilities are yet to be established. 
 
Table 1.
 
Diagnosis of Studied Eyes and Histologic Classification of CNV
Table 1.
 
Diagnosis of Studied Eyes and Histologic Classification of CNV
Specimen Age/ Sex Eye Clinical Description Histologic Classification CNV
Subretinal/Sub-RPE FV/FC Other Characteristics
Co1 83/M L Corneal ulcer
Co2 57/M L Recurrent conjunctival melanoma
Co3 42/M L Choroidal melanoma
Co4 69/F R Orbital metastasis, after irradiation
DM1 34/M L PDR
DM2 63/F R PDR
DM3 75/F R PDR
CNV1 79/M U Surgically excised CNV Mixed FV + FC Hemorrhage
CNV2 79/F U Surgically excised CNV Subretinal FV + FC Hemorrhage
CNV3 76/F R Surgically excised CNV Subretinal FV BLD
CNV4 79/M U Surgically excised CNV Mixed FV + FC BLD, hemorrhage
CNV5 78/M U Surgically excised CNV Sub-RPE FV + FC BLD, confluent soft drusen
CNV6 72/M L Disciform MD Mixed FV + FC BLD, hemorrhage
CNV7 86/M L Disciform MD, acute glaucoma Sub-RPE FV + FC BLD, hemorrhage, retinal detachment, posterior uveitis
CNV8 91/M L Disciform MD, donor eye Mixed FC Disciform MD, BLD
CNV9 87/M L Disciform MD, donor eye Mixed FV + FC Disciform MD, BLD
CNV10 83/M R Painful eye, suspected uveal melanoma Mixed FV + FC Ischemic retinal disease, disciform MD, BLD, hemorrhage
CNV11 73/M L Disciform MD Subretinal FV + FC
CNV12 73/M R Disciform MD, after irradiation Subretinal FV
CNV13 82/M R Disciform MD Mixed FC Confluent soft drusen
CNV14 80/F L Disciform MD, hemorrhage, secondary glaucoma Mixed FV + FC BLD, hemorrhage
Table 2.
 
Expression of IGF-I and IGF Receptor Type 1 in CNV
Table 2.
 
Expression of IGF-I and IGF Receptor Type 1 in CNV
Specimen IGF-I mRNA IGF-IR mRNA IGF-IR Protein
EC RP FB O EC RP FB O EC RP FB O
CNV1 0 0 0 1 2 2 2 2 1 1 1 1
CNV2 0 0 0 0 2 2 2 2 1 2 2 1
CNV3 1 2 1 1 2 1 1 2 1 1 0 0
CNV4 1 2 1 1 NC NC NC NC 1 2 0 0
CNV5 1 1 0 1 1 2 1 1 1 2 NC NC
CNV6 0 0 0 0 1 1 1 0 1 1 0 1
CNV7 NC 0 NC 1 1 2 1 1 0 1 0 0
CNV8 0 0 0 0 0 0 0 0 0 1 1 0
CNV9 2 2 1 1 2 2 2 2 NC NC NC NC
CNV10 1 1 1 1 1 1 1 0 1 1 1 0
CNV11 0 1 1 NC 2 2 2 2 1 1 0 1
CNV12 0 1 1 1 1 1 1 1 2 2 1 1
CNV13 0 0 0 0 0 0 0 0 0 1 0 0
CNV14 0 1 0 1 1 1 1 1 0 1 0 0
Figure 1.
 
Localization of IGF-I and IGF-IR in the human eye and in eyes with diabetic proliferative retinopathy. Expression of IGF-I mRNA (A, D, G) and IGF-IR mRNA (B, E, H) in paraffin-embedded tissue, detected by in situ hybridization with digoxigenin-labeled probes, stained with NBT/BCIP (blue), and counterstained with nuclear red. Expression of IGF-IR protein (C, F, I) in paraffin-embedded tissue detected with polyclonal antibodies and visualized with an alkaline phosphatase system, using a red chromogen and hematoxylin counterstaining. Short arrows: capillary endothelial cells; long arrows: RPE cells. (AF) Posterior pole of normal eye (Co1) with expression of IGF-I (A, D) and IGF-IR (B, E) mRNA and IGF-IR protein (C, F). (G, H, I) Diabetic preretinal membrane (DM2) with expression of IGF-I (G) and IGF-IR (H) mRNA and IGF-IR protein (I). Original magnification, ×400.
Figure 1.
 
Localization of IGF-I and IGF-IR in the human eye and in eyes with diabetic proliferative retinopathy. Expression of IGF-I mRNA (A, D, G) and IGF-IR mRNA (B, E, H) in paraffin-embedded tissue, detected by in situ hybridization with digoxigenin-labeled probes, stained with NBT/BCIP (blue), and counterstained with nuclear red. Expression of IGF-IR protein (C, F, I) in paraffin-embedded tissue detected with polyclonal antibodies and visualized with an alkaline phosphatase system, using a red chromogen and hematoxylin counterstaining. Short arrows: capillary endothelial cells; long arrows: RPE cells. (AF) Posterior pole of normal eye (Co1) with expression of IGF-I (A, D) and IGF-IR (B, E) mRNA and IGF-IR protein (C, F). (G, H, I) Diabetic preretinal membrane (DM2) with expression of IGF-I (G) and IGF-IR (H) mRNA and IGF-IR protein (I). Original magnification, ×400.
Figure 2.
 
Localization of IGF-I and IGF-IR in eyes with neovascular AMD. Expression of IGF-I mRNA (A, D, G, J, L), IGF-IR mRNA (B, E, H, K, M) in paraffin-embedded tissue, detected by in situ hybridization with digoxigenin-labeled probes, colored with NBT/BCIP (blue), and counterstained with nuclear red. Expression of IGF-IR protein (C, F, I) in paraffin-embedded tissue detected with polyclonal antibodies and visualized with an alkaline phosphatase system, using a red chromogen and hematoxylin counterstaining. Short arrows: capillary endothelial cells, long arrows: RPE. Surgically excised specimens of CNV: CNV2 with expression of IGF-I (A) and IGF-IR (B) mRNA and IGF-IR protein (C); CNV3 with expression of IGF-I (D) and IGF-IR (E) mRNA and IGF-IR protein (F); sub-RPE CNV11 with expression of IGF-I (G) and IGF-IR (H) mRNA and IGF-IR protein (I); subretinal CNVg with expression of IGF-I (J) and IGF-IR (K) mRNA. Protein detection of IGF-IR with immunohistochemistry produced aspecific staining and is therefore not shown. Expression of sense mRNA probes of IGF-I (L) and IGF-IR (M) in subretinal CNV (CNV9). Original magnification, ×400.
Figure 2.
 
Localization of IGF-I and IGF-IR in eyes with neovascular AMD. Expression of IGF-I mRNA (A, D, G, J, L), IGF-IR mRNA (B, E, H, K, M) in paraffin-embedded tissue, detected by in situ hybridization with digoxigenin-labeled probes, colored with NBT/BCIP (blue), and counterstained with nuclear red. Expression of IGF-IR protein (C, F, I) in paraffin-embedded tissue detected with polyclonal antibodies and visualized with an alkaline phosphatase system, using a red chromogen and hematoxylin counterstaining. Short arrows: capillary endothelial cells, long arrows: RPE. Surgically excised specimens of CNV: CNV2 with expression of IGF-I (A) and IGF-IR (B) mRNA and IGF-IR protein (C); CNV3 with expression of IGF-I (D) and IGF-IR (E) mRNA and IGF-IR protein (F); sub-RPE CNV11 with expression of IGF-I (G) and IGF-IR (H) mRNA and IGF-IR protein (I); subretinal CNVg with expression of IGF-I (J) and IGF-IR (K) mRNA. Protein detection of IGF-IR with immunohistochemistry produced aspecific staining and is therefore not shown. Expression of sense mRNA probes of IGF-I (L) and IGF-IR (M) in subretinal CNV (CNV9). Original magnification, ×400.
Young, RW. (1987) Pathophysiology of age-related macular degeneration Surv Ophthalmol 31,291-306 [CrossRef] [PubMed]
D’Amore, PA. (1994) Mechanisms of retinal and choroidal neovascularization Invest Ophthalmol Vis Sci 35,3974-3979 [PubMed]
Kwak, N, Okamoto, N, Wood, JM, Campochiaro, PA. (2000) VEGF is major stimulator in model of choroidal neovascularization Invest Ophthalmol Vis Sci 41,3158-3164 [PubMed]
Kliffen, M, Sharma, HS, Mooy, CM, Kerkvliet, S, de Jong, PT. (1997) Increased expression of angiogenic growth factors in age-related maculopathy Br J Ophthalmol 81,154-162 [CrossRef] [PubMed]
Lambooij, AC, Kuijpers, RW, van Lichtenauer-Kaligis, EG, et al (2000) Somatostatin receptor 2A expression in choroidal neovascularization secondary to age-related macular degeneration Invest Ophthalmol Vis Sci 41,2329-2335 [PubMed]
Grant, MB, Caballero, S, Millard, WJ. (1993) Inhibition of IGF-I and b-FGF stimulated growth of human retinal endothelial cells by the somatostatin analogue, octreotide: a potential treatment for ocular neovascularization Regul Pept 48,267-278 [CrossRef] [PubMed]
Spraul, CW, Baldysiak-Figiel, A, Lang, GK, Lang, GE. (2002) Octreotide inhibits growth factor-induced bovine choriocapillary endothelial cells in vitro Graefes Arch Clin Exp Ophthalmol 240,227-231 [CrossRef] [PubMed]
Smith, LE, Kopchick, JJ, Chen, W, et al (1997) Essential role of growth hormone in ischemia-induced retinal neovascularization Science 276,1706-1709 [CrossRef] [PubMed]
King, GL, Goodman, AD, Buzney, S, Moses, A, Kahn, CR. (1985) Receptors and growth-promoting effects of insulin and insulinlike growth factors on cells from bovine retinal capillaries and aorta J Clin Invest 75,1028-1036 [CrossRef] [PubMed]
Kaven, CW, Spraul, CW, Zavazava, NK, Lang, GK, Lang, GE. (2000) Growth factor combinations modulate human retinal pigment epithelial cell proliferation Curr Eye Res 20,480-487 [CrossRef] [PubMed]
Spraul, CW, Kaven, C, Amann, J, Lang, GK, Lang, GE. (2000) Effect of insulin-like growth factors 1 and 2, and glucose on the migration and proliferation of bovine retinal pigment epithelial cells in vitro Ophthalmic Res 32,244-248 [CrossRef] [PubMed]
Hernandez-Sanchez, C, Lopez-Carranza, A, Alarcon, C, de La Rosa, EJ, de Pablo, F. (1995) Autocrine/paracrine role of insulin-related growth factors in neurogenesis: local expression and effects on cell proliferation and differentiation in retina Proc Natl Acad Sci USA 92,9834-9838 [CrossRef] [PubMed]
Punglia, RS, Lu, M, Hsu, J, et al (1997) Regulation of vascular endothelial growth factor expression by insulin-like growth factor I Diabetes 46,1619-1626 [CrossRef] [PubMed]
Martin, DM, Yee, D, Feldman, EL. (1992) Gene expression of the insulin-like growth factors and their receptors in cultured human retinal pigment epithelial cells Brain Res Mol Brain Res 12,181-186 [CrossRef] [PubMed]
Kociok, N, Heppekausen, H, Schraermeyer, U, et al (1998) The mRNA expression of cytokines and their receptors in cultured iris pigment epithelial cells: a comparison with retinal pigment epithelial cells Exp Eye Res 67,237-250 [CrossRef] [PubMed]
Takagi, H, Yoshimura, N, Tanihara, H, Honda, Y. (1994) Insulin-like growth factor-related genes, receptors, and binding proteins in cultured human retinal pigment epithelial cells Invest Ophthalmol Vis Sci 35,916-923 [PubMed]
Spoerri, PE, Ellis, EA, Tarnuzzer, RW, Grant, MB. (1998) Insulin-like growth factor: receptor and binding proteins in human retinal endothelial cell cultures of diabetic and non-diabetic origin Growth Horm IGF Res 8,125-132 [CrossRef] [PubMed]
Ulbig, MW, Wolfensberger, TJ, Hiscott, P, Ationu, A, Carter, ND, Gregor, ZJ. (1995) Insulin-like growth factor I (IGF-I) receptor/binding protein in human diabetic epiretinal membranes Ger J Ophthalmol 4,264-268 [PubMed]
Gerhardinger, C, McClure, KD, Romeo, G, Podesta, F, Lorenzi, M. (2001) IGF-I mRNA and signaling in the diabetic retina Diabetes 50,175-183 [CrossRef] [PubMed]
Jansen, M, van Schaik, FM, Ricker, AT, et al (1983) Sequence of cDNA encoding human insulin-like growth factor I precursor Nature 306,609-611 [CrossRef] [PubMed]
Burren, CP, Berka, JL, Edmondson, SR, Werther, GA, Batch, JA. (1996) Localization of mRNAs for insulin-like growth factor-I (IGF-I), IGF-I receptor, and IGF binding proteins in rat eye Invest Ophthalmol Vis Sci 37,1459-1468 [PubMed]
Ocrant, I, Valentino, KL, King, MG, Wimpy, TH, Rosenfeld, RG, Baskin, DG. (1989) Localization and structural characterization of insulin-like growth factor receptors in mammalian retina Endocrinology 125,2407-2413 [CrossRef] [PubMed]
Grant, M, King, GL. (1995) IGF-I and blood vessels: Implications for microvascular and macrovascular disease Diabetes Rev 3,113-128
Politi, LE, Rotstein, NP, Salvador, G, Giusto, NM, Insua, MF. (2001) Insulin-like growth factor-I is a potential trophic factor for amacrine cells J Neurochem 76,1199-1211 [CrossRef] [PubMed]
Seigel, GM, Chiu, L, Paxhia, A. (2000) Inhibition of neuroretinal cell death by insulin-like growth factor-1 and its analogs Mol Vis 6,157-163 [PubMed]
Kermer, P, Klocker, N, Labes, M, Bahr, M. (2000) Insulin-like growth factor-I protects axotomized rat retinal ganglion cells from secondary death via PI3-K-dependent Akt phosphorylation and inhibition of caspase-3 in vivo J Neurosci 20,2-8 [PubMed]
Waldbillig, RJ, Pfeffer, BA, Schoen, TJ, et al (1991) Evidence for an insulin-like growth factor autocrine-paracrine system in the retinal photoreceptor-pigment epithelial cell complex J Neurochem 57,1522-1533 [CrossRef] [PubMed]
Lowe, WL, Jr, Florkiewicz, RZ, Yorek, MA, Spanheimer, RG, Albrecht, BN. (1995) Regulation of growth factor mRNA levels in the eyes of diabetic rats Metabolism 44,1038-1045 [CrossRef] [PubMed]
Meyer-Schwickerath, R, Pfeiffer, A, Blum, WF, et al (1993) Vitreous levels of the insulin-like growth factors I and II, and the insulin-like growth factor binding proteins 2 and 3, increase in neovascular eye disease: studies in nondiabetic and diabetic subjects J Clin Invest 92,2620-2625 [CrossRef] [PubMed]
Grant, M, Russell, B, Fitzgerald, C, Merimee, TJ. (1986) Insulin-like growth factors in vitreous: studies in control and diabetic subjects with neovascularization Diabetes 35,416-420 [CrossRef] [PubMed]
Boulton, M, Gregor, Z, McLeod, D, et al (1997) Intravitreal growth factors in proliferative diabetic retinopathy: correlation with neovascular activity and glycaemic management Br J Ophthalmol 81,228-233 [CrossRef] [PubMed]
Burgos, R, Mateo, C, Canton, A, Hernandez, C, Mesa, J, Simo, R. (2000) Vitreous levels of IGF-I, IGF binding protein 1, and IGF binding protein 3 in proliferative diabetic retinopathy: a case-control study Diabetes Care 23,80-83 [CrossRef] [PubMed]
Spranger, J, Buhnen, J, Jansen, V, et al (2000) Systemic levels contribute significantly to increased intraocular IGF- I, IGF-II and IGF-BP3 in proliferative diabetic retinopathy Horm Metab Res 32,196-200 [CrossRef] [PubMed]
Grant, MB, Mames, RN, Fitzgerald, C, Ellis, EA, Aboufriekha, M, Guy, J. (1993) Insulin-like growth factor I acts as an angiogenic agent in rabbit cornea and retina: comparative studies with basic fibroblast growth factor Diabetologia 36,282-291 [CrossRef] [PubMed]
Danis, RP, Bingaman, DP. (1997) Insulin-like growth factor-1 retinal microangiopathy in the pig eye Ophthalmology 104,1661-1669 [CrossRef] [PubMed]
Smith, LE, Shen, W, Perruzzi, C, et al (1999) Regulation of vascular endothelial growth factor-dependent retinal neovascularization by insulin-like growth factor-1 receptor Nat Med 5,1390-1395 [CrossRef] [PubMed]
Colas, B, Cambillau, C, Buscail, L, et al (1992) Stimulation of a membrane tyrosine phosphatase activity by somatostatin analogues in rat pancreatic acinar cells Eur J Biochem 207,1017-1024 [CrossRef] [PubMed]
Klisovic, DD, Sall, JW, O’Dorisio, MS, Katz, SE. (2001) Somatostatin and octreotide inhibit IGF-1 mediated tyrosine phosphorylation of IGF-1Rbeta and induction of VEGF gene expression in human RPE cells [ARVO Abstract] Invest Ophthalmol Vis Sci 42,S244Abstract nr 1316
LeRoith, D, Clemmons, D, Nissley, P, Rechler, MM. (1992) NIH conference. Insulin-like growth factors in health and disease Ann Intern Med 116,854-862 [CrossRef] [PubMed]
Gartner, MH, Benson, JD, Caldwell, MD. (1992) Insulin-like growth factors I and II expression in the healing wound J Surg Res 52,389-394 [CrossRef] [PubMed]
Figure 1.
 
Localization of IGF-I and IGF-IR in the human eye and in eyes with diabetic proliferative retinopathy. Expression of IGF-I mRNA (A, D, G) and IGF-IR mRNA (B, E, H) in paraffin-embedded tissue, detected by in situ hybridization with digoxigenin-labeled probes, stained with NBT/BCIP (blue), and counterstained with nuclear red. Expression of IGF-IR protein (C, F, I) in paraffin-embedded tissue detected with polyclonal antibodies and visualized with an alkaline phosphatase system, using a red chromogen and hematoxylin counterstaining. Short arrows: capillary endothelial cells; long arrows: RPE cells. (AF) Posterior pole of normal eye (Co1) with expression of IGF-I (A, D) and IGF-IR (B, E) mRNA and IGF-IR protein (C, F). (G, H, I) Diabetic preretinal membrane (DM2) with expression of IGF-I (G) and IGF-IR (H) mRNA and IGF-IR protein (I). Original magnification, ×400.
Figure 1.
 
Localization of IGF-I and IGF-IR in the human eye and in eyes with diabetic proliferative retinopathy. Expression of IGF-I mRNA (A, D, G) and IGF-IR mRNA (B, E, H) in paraffin-embedded tissue, detected by in situ hybridization with digoxigenin-labeled probes, stained with NBT/BCIP (blue), and counterstained with nuclear red. Expression of IGF-IR protein (C, F, I) in paraffin-embedded tissue detected with polyclonal antibodies and visualized with an alkaline phosphatase system, using a red chromogen and hematoxylin counterstaining. Short arrows: capillary endothelial cells; long arrows: RPE cells. (AF) Posterior pole of normal eye (Co1) with expression of IGF-I (A, D) and IGF-IR (B, E) mRNA and IGF-IR protein (C, F). (G, H, I) Diabetic preretinal membrane (DM2) with expression of IGF-I (G) and IGF-IR (H) mRNA and IGF-IR protein (I). Original magnification, ×400.
Figure 2.
 
Localization of IGF-I and IGF-IR in eyes with neovascular AMD. Expression of IGF-I mRNA (A, D, G, J, L), IGF-IR mRNA (B, E, H, K, M) in paraffin-embedded tissue, detected by in situ hybridization with digoxigenin-labeled probes, colored with NBT/BCIP (blue), and counterstained with nuclear red. Expression of IGF-IR protein (C, F, I) in paraffin-embedded tissue detected with polyclonal antibodies and visualized with an alkaline phosphatase system, using a red chromogen and hematoxylin counterstaining. Short arrows: capillary endothelial cells, long arrows: RPE. Surgically excised specimens of CNV: CNV2 with expression of IGF-I (A) and IGF-IR (B) mRNA and IGF-IR protein (C); CNV3 with expression of IGF-I (D) and IGF-IR (E) mRNA and IGF-IR protein (F); sub-RPE CNV11 with expression of IGF-I (G) and IGF-IR (H) mRNA and IGF-IR protein (I); subretinal CNVg with expression of IGF-I (J) and IGF-IR (K) mRNA. Protein detection of IGF-IR with immunohistochemistry produced aspecific staining and is therefore not shown. Expression of sense mRNA probes of IGF-I (L) and IGF-IR (M) in subretinal CNV (CNV9). Original magnification, ×400.
Figure 2.
 
Localization of IGF-I and IGF-IR in eyes with neovascular AMD. Expression of IGF-I mRNA (A, D, G, J, L), IGF-IR mRNA (B, E, H, K, M) in paraffin-embedded tissue, detected by in situ hybridization with digoxigenin-labeled probes, colored with NBT/BCIP (blue), and counterstained with nuclear red. Expression of IGF-IR protein (C, F, I) in paraffin-embedded tissue detected with polyclonal antibodies and visualized with an alkaline phosphatase system, using a red chromogen and hematoxylin counterstaining. Short arrows: capillary endothelial cells, long arrows: RPE. Surgically excised specimens of CNV: CNV2 with expression of IGF-I (A) and IGF-IR (B) mRNA and IGF-IR protein (C); CNV3 with expression of IGF-I (D) and IGF-IR (E) mRNA and IGF-IR protein (F); sub-RPE CNV11 with expression of IGF-I (G) and IGF-IR (H) mRNA and IGF-IR protein (I); subretinal CNVg with expression of IGF-I (J) and IGF-IR (K) mRNA. Protein detection of IGF-IR with immunohistochemistry produced aspecific staining and is therefore not shown. Expression of sense mRNA probes of IGF-I (L) and IGF-IR (M) in subretinal CNV (CNV9). Original magnification, ×400.
Table 1.
 
Diagnosis of Studied Eyes and Histologic Classification of CNV
Table 1.
 
Diagnosis of Studied Eyes and Histologic Classification of CNV
Specimen Age/ Sex Eye Clinical Description Histologic Classification CNV
Subretinal/Sub-RPE FV/FC Other Characteristics
Co1 83/M L Corneal ulcer
Co2 57/M L Recurrent conjunctival melanoma
Co3 42/M L Choroidal melanoma
Co4 69/F R Orbital metastasis, after irradiation
DM1 34/M L PDR
DM2 63/F R PDR
DM3 75/F R PDR
CNV1 79/M U Surgically excised CNV Mixed FV + FC Hemorrhage
CNV2 79/F U Surgically excised CNV Subretinal FV + FC Hemorrhage
CNV3 76/F R Surgically excised CNV Subretinal FV BLD
CNV4 79/M U Surgically excised CNV Mixed FV + FC BLD, hemorrhage
CNV5 78/M U Surgically excised CNV Sub-RPE FV + FC BLD, confluent soft drusen
CNV6 72/M L Disciform MD Mixed FV + FC BLD, hemorrhage
CNV7 86/M L Disciform MD, acute glaucoma Sub-RPE FV + FC BLD, hemorrhage, retinal detachment, posterior uveitis
CNV8 91/M L Disciform MD, donor eye Mixed FC Disciform MD, BLD
CNV9 87/M L Disciform MD, donor eye Mixed FV + FC Disciform MD, BLD
CNV10 83/M R Painful eye, suspected uveal melanoma Mixed FV + FC Ischemic retinal disease, disciform MD, BLD, hemorrhage
CNV11 73/M L Disciform MD Subretinal FV + FC
CNV12 73/M R Disciform MD, after irradiation Subretinal FV
CNV13 82/M R Disciform MD Mixed FC Confluent soft drusen
CNV14 80/F L Disciform MD, hemorrhage, secondary glaucoma Mixed FV + FC BLD, hemorrhage
Table 2.
 
Expression of IGF-I and IGF Receptor Type 1 in CNV
Table 2.
 
Expression of IGF-I and IGF Receptor Type 1 in CNV
Specimen IGF-I mRNA IGF-IR mRNA IGF-IR Protein
EC RP FB O EC RP FB O EC RP FB O
CNV1 0 0 0 1 2 2 2 2 1 1 1 1
CNV2 0 0 0 0 2 2 2 2 1 2 2 1
CNV3 1 2 1 1 2 1 1 2 1 1 0 0
CNV4 1 2 1 1 NC NC NC NC 1 2 0 0
CNV5 1 1 0 1 1 2 1 1 1 2 NC NC
CNV6 0 0 0 0 1 1 1 0 1 1 0 1
CNV7 NC 0 NC 1 1 2 1 1 0 1 0 0
CNV8 0 0 0 0 0 0 0 0 0 1 1 0
CNV9 2 2 1 1 2 2 2 2 NC NC NC NC
CNV10 1 1 1 1 1 1 1 0 1 1 1 0
CNV11 0 1 1 NC 2 2 2 2 1 1 0 1
CNV12 0 1 1 1 1 1 1 1 2 2 1 1
CNV13 0 0 0 0 0 0 0 0 0 1 0 0
CNV14 0 1 0 1 1 1 1 1 0 1 0 0
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