Autocrine effects of IGF-I-induced VEGF and IGFBP-3 secretion in retinal pigment epithelial cell line ARPE-19

Mark G. Slomiany and Steven A. Rosenzweig

Department of Cell and Molecular Pharmacology and Experimental Therapeutics and Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina 29403

Submitted 15 December 2003 ; accepted in final form 5 May 2004


    ABSTRACT
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hypoxia-induced physiological stress plays a central role in various neovascular diseases of the eye. Increased expression of hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) and subsequent formation of HIF-1 dimers active at the vascular endothelial growth factor (VEGF) promoter lead to expression of this potent angiogenic factor in the retina, including retinal pigment epithelial (RPE) cells. We previously demonstrated that insulin-like growth factor I (IGF-I) stimulates VEGF and IGF binding protein (IGFBP)-3 secretion in RPE cells. In this study we examined IGF-I-induced HIF-1{alpha} expression, VEGF and IGFBP-3 secretion, and the autocrine actions of VEGF and IGFBP-3 on these processes in the spontaneously transformed RPE cell line ARPE-19. Cells were treated with CoCl2, IGF-I, recombinant human (rh)IGFBP-3, and rhVEGF. Immunoblot analysis revealed IGF-I-induced upregulation of total HIF-1{alpha} protein, whereas luciferase reporter assays of HIF-1 transcriptional activity demonstrated accumulation of HIF-1{alpha} correlated with the formation of functional HIF-1 heterodimers. Western and ligand blot analyses of RPE cell conditioned medium confirmed that IGF-I stimulated VEGF and IGFBP-3 secretion. rhVEGF stimulated IGFBP-3 secretion in an IGF-I- and HIF-1{alpha}-independent manner, whereas rhIGFBP-3 attenuated IGF-I-induced VEGF secretion. These findings demonstrate the multifaceted autocrine regulation of IGF-I-induced VEGF secretion by IGFBP-3 secreted in response to both IGF-I and, to a lesser extent, VEGF. These results provide evidence for HIF-1-dependent and -independent mechanisms by which IGF-I regulates VEGF and IGFBP-3 secretion.

angiogenesis; hypoxia-inducible factor 1; hypoxia; insulin-like growth factor binding protein; vascular endothelial growth factor


ADAPTATION TO HYPOXIA involves changes in the protein expression and DNA binding activity of the hypoxia-inducible factors (HIFs), the most prevalent isoform being HIF-1. This family of transcription factors consists of an {alpha}-subunit, whose proteasomal degradation and thus relative abundance are regulated by both cytokines and oxygen tension, and a constitutively expressed {beta}-subunit. Binding of HIF-1 to the hypoxia response element (HRE) of the vascular endothelial growth factor (VEGF) promoter results in transcriptional activity (39, 44).

VEGF is a potent angiogenic and permeability-enhancing peptide causally linked to neovascularization of the retina and iris (1, 2, 26, 29, 47). In choroidal neovascularization (CNV), a condition that develops in 10% of age-related macular degeneration (AMD) sufferers, newly formed choroidal blood vessels enter the subretinal space, where leakage and bleeding lead to retinal detachment and photoreceptor death (10, 1820, 32, 37). The ineffectiveness of the principal treatments for CNV, namely argon laser photocoagulation and photodynamic therapy, underscores the reason why this disease represents the most common cause of severe vision loss in elderly patients in developed countries (31, 32). Identification of the mediators of ocular angiogenesis would provide important targets for the development of selective inhibitors of CNV (23, 31). Of particular interest to our studies is the retinal pigment epithelium (RPE), a monolayer of highly specialized epithelial cells interposed between the retinal photoreceptors and the choroid (6, 68). Central to photoreceptor survival and function, the RPE is the major source of angiogenic (e.g., VEGF) and antiangiogenic [e.g., pigment epithelium-derived factor (PEDF)] factors and may therefore play a central role in the modulation and progression of CNV (4, 11, 24, 28, 30, 40, 56, 62, 65).

A number of animal models support a role for increased RPE VEGF secretion in the progression of CNV (9, 27, 50). In addition to elevated VEGF levels in the vitreous (62), the RPE and surrounding subretinal membranes express increased levels of VEGF and its receptor kinase insert domain receptor (KDR)/fetal liver kinase receptor-1 (Flk-1) in CNV (3, 48, 58); these increased levels have been attributed to the cellular hypoxic response (59). A number of factors regulate VEGF production; among them, insulin-like growth factor (IGF)-I has been demonstrated to stimulate VEGF expression. Punglia and coworkers (40) showed that increased serum and vitreous IGF-I levels correlate with a wide variety of ischemic retinal disorders linked to neovascularization of the retina and iris. Examination of dissected postmortem RPE-choroid as well as cultured RPE cell lines has found transcription and cell membrane localization of the IGF-I and IGF-II receptors (34, 38, 53, 54, 59, 63) as well as transcription and secretion of IGF-I and IGF-II (34, 36, 38, 53, 63), along with IGF binding proteins (IGFBPs) 1–6 and the IGFBP-related protein IGFBP-rP1 (36, 38, 53, 63, 64). Because IGFs bind with higher affinity to IGFBPs than to the IGF-I receptor, IGFBPs are capable of acting as antagonists by reducing IGF bioavailability through sequestration (25, 42). Thus the RPE provides the necessary components for a subretinal autocrine-paracrine IGF system capable of modulating retinal function as well as contributing to the pathogenesis of CNV (60, 67).

On the basis of a growing body of evidence demonstrating that IGF-I can induce HIF-1 activity and the secretion of VEGF and IGFBP-3 in RPE cells in vivo and in vitro (15–17, 22, 35, 41, 43, 45), we used the spontaneously transformed RPE cell line ARPE-19 (12) to examine the effect of IGF-I on HIF-1{alpha} protein expression, VEGF and IGFBP-3 secretion, and the autocrine effects of VEGF and IGFBP-3. Immunoblot analysis revealed IGF-I-induced upregulation of total HIF-1{alpha} protein, whereas luciferase reporter assays of HIF-1 transcriptional activity demonstrated accumulation of HIF-1{alpha} correlated with the formation of functional HIF-1 heterodimers. In contrast, addition of exogenous VEGF had no significant effect on HIF-1{alpha} protein levels in control or IGF-I-stimulated cells. Western and ligand blot analyses of conditioned medium confirmed that IGF-I induced VEGF and IGFBP-3 secretion, recombinant human (rh)VEGF induced IGFBP-3 secretion, and rhIGFBP-3 attenuated IGF-I-stimulated VEGF release. These findings demonstrate that, as seen for VEGF, IGF-I-induced stimulation of IGFBP-3 secretion in RPE cells correlates with increased HIF-1{alpha} expression and nuclear localization. We have also identified a unique autocrine function of VEGF in inducing the secretion of IGFBP-3 in control and IGF-I-stimulated ARPE-19 cells without affecting HIF-1 protein expression. Finally, our study demonstrates the negative-feedback role of IGFBP-3 in sequestering and thereby attenuating IGF-I-induced VEGF secretion.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials and reagents. ARPE-19 cells were obtained from the American Type Culture Collection (Manassas, VA). Fetal bovine serum (FBS) was purchased from Atlas Biologicals (Fort Collins, CO). DMEM was purchased from Sigma (St. Louis, MO). IGF-I and rhVEGF (bacterial origin) were generously provided by Genentech (San Francisco, CA). rhIGFBP-3 (N109D, bacterial origin) was obtained from Upstate (Lake Placid, NY). CoCl2 was from Fisher Scientific (Fair Lawn, NJ). HIF-1{alpha} monoclonal antibody was obtained from Transduction Laboratories (Lexington, KY), HIF-1{beta} monoclonal antibody from Novus (Littleton, CO), and {beta}-actin polyclonal antibody from Sigma. VEGF polyclonal antibody and horseradish peroxidase (HRP) were purchased from Chemicon (Temecula, CA). Neutravidin-HRP and bicinchoninic acid (BCA) reagent were obtained from Pierce (Rockford, IL). Tunicamycin was purchased from Calbiochem (San Diego, CA). ECL reagent was obtained from Amersham Biosciences (Clearbrook, IL) and Biomax film from Kodak (Rochester, NY). Fugene 6 was obtained from Roche. The Dual-Luciferase Reporter Assay System was purchased from Promega. p2.1 was a generous gift from the laboratory of Dr. Gregg L. Semenza of the Johns Hopkins University School of Medicine (Baltimore, MD), and SV40 Renilla, obtained from Promega, was provided by Dr. D. T. Kurtz, Medical University of South Carolina. All other chemicals were of reagent grade or higher.

Tissue culture. ARPE-19 cells were incubated in a 1-to-1 ratio of Dulbecco's modified Eagle's medium Base D-5030 and Nutrient Mixture F-12 (Ham) N-6760 with 10% FBS and 10 µl/ml penicillin-streptomycin solution. Unless otherwise stated, cells were maintained at 37°C in a humidified 5% CO2-95% air incubator.

IGF-I, IGFBP-3, CoCl2, tunicamycin, and VEGF treatments. ARPE-19 cells were seeded at a density of 8.6 x 105/well in six-well (9.6-cm2 area) plates. Confluent cells were serum starved (FBS was eliminated in all experiments) for 24 h, to remove known stimulatory growth factors (including IGF-I), before the indicated treatment in fresh, serum-free, medium.

Immunoblot and ligand blot analysis. Confluent serum-starved cells were treated with IGF-I or CoCl2 as indicated, and whole cell lysates were prepared with a modified RIPA buffer containing (in mM) 50 Tris·HCl pH 7.4, 150 NaCl, 10 EDTA, 1 PMSF, 2 sodium orthovanadate, and 10 NaF with 1% Triton X-100 and 10 µg/ml aprotinin and leupeptin. Protein content was determined by BCA assay (Pierce), and 100-µg aliquots were solubilized in SDS sample buffer. VEGF and IGFBP-3 in conditioned medium were quantified after precipitation in 10% trichloroacetic acid (TCA), washing of the pellet with acetone, and solubilization in SDS sample buffer. Proteins so collected were resolved on 12.5% nonreducing polyacrylamide gels, transferred to nitrocellulose (Osmonics, Westborough MA) with a TE-70 SemiPhor apparatus (Hoefer Scientific Instruments, San Francisco, CA), and subjected to ligand or immunoblot analysis. For ligand blot analysis, protein-containing nitrocellulose membranes were washed for 10 min at 23°C in Tris-buffered saline (TBS) containing 3% Triton X-100 and blocked for 1 h with TBS containing 0.2% gelatin. Blots were probed overnight at 4°C with 10 ng/ml tetrabiotinylated IGF-I (Robinson SA and Rosenzweig SA, unpublished data), followed by a 2-h incubation at 23°C with 200 ng/ml Neutravidin-HRP in TBS containing 0.1% Tween 20 and 0.1% BSA. Blots were visualized with the ECL reagent (Amersham Biosciences) on Biomax film (Kodak). Films were subsequently digitized to tiff format, and band intensity was quantified with NIH Image, version II.

For immunoblots, nitrocellulose membranes were blocked for 1 h in bovine lacto transfer technique optimizer (BLOTTO), a TBS solution containing 0.1% Tween 20 and 5% milk protein (reviewed in Ref. 51), before being probed with 1 µg/ml VEGF polyclonal antibody or 1 µg/ml HIF-1{alpha} monoclonal antibody, 1 µg/ml HIF-1{beta} monoclonal antibody, or 1:10,000 {beta}-actin monoclonal antibody in BLOTTO. HRP-linked secondary antibodies diluted 1:5,000 in BLOTTO were subsequently added for 2 h. To reprobe HIF-1{alpha} immunoblots for HIF-1{beta} or {beta}-actin levels, antibodies were removed from the nitrocellulose via the application of Chemicon light stripping solution according to the manufacturer's instructions. Blots were visualized with the ECL reagent as described above.

Luciferase assays. To assay the transcriptional activity of HIF-1, we used the pGL2 basic p2.1 enolase 1 (ENO1) promoter vector, which contains a 68-bp ENO1 promoter fragment encompassing a HIF-1 binding site downstream from the luciferase gene (46). Each well of subconfluent ARPE-19 cells was transiently cotransfected with 100 ng of reporter plasmid and 50 ng of pRL-SV40 Renilla as a control for transfection efficiency. After 24 h, cells were treated with 100 nM IGF-I or 100 µM CoCl2 in 500 µl/well fresh serum-free medium. After an 18-h incubation, cells were lysed in 100 µl/well passive lysis buffer provided with the Dual-Luciferase Reporter Assay System. Cells were scraped and centrifuged for 10 min at 18,890 g, and 20 µl of supernatant per sample was loaded on a 96-well plate and processed for luciferase activity on the Victor2 1420 Multilabel Counter (PerkinElmer Life Sciences, Downers Grove, IL) with the firefly and Renilla luciferase buffers provided with the Dual-Luciferase kit.


    RESULTS
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
HIF-1{alpha} expression and activity. To examine the effect of IGF-I on HIF-1{alpha} protein expression, serum-starved ARPE-19 cells were exposed to a stimulatory dose of IGF-I (100 nM) for 4 h. This treatment resulted in increased HIF-1{alpha} protein levels based on immunoblot analysis (Fig. 1A) and normalization to HIF-1{beta} control levels (Fig. 1, B and C). As a positive control, cells were exposed to 100 µM CoCl2, a chemical hypoxia-inducing agent that abrogates HIF-1{alpha} degradation (14). The blots were stripped and reprobed for HIF-1{beta}, the constitutively expressed HIF-1{alpha} binding partner present in the HIF-1 heterodimer (Fig. 1B). As expected, neither IGF-I nor CoCl2 induced significant changes in HIF-1{beta} expression in ARPE-19 cells, demonstrating a specific effect of these agents on HIF-1{alpha} expression. To establish the connection between HIF-1{alpha} protein expression and functional HIF-1 heterodimer formation, transcriptional activity was analyzed (Fig. 1D). ARPE-19 cells were cotransfected with the p2.1 HIF-1 reporter plasmid and the pRL-SV40 control plasmid and subjected to the conditions described above for 18 h. As expected, IGF-I- and CoCl2-stimulated increases in HIF-1 transcriptional activity were consistent with increases in HIF-1{alpha} protein expression.



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Fig. 1. Effect of insulin-like growth factor (IGF)-I and CoCl2 treatment on hypoxia-inducible factor (HIF)-1{alpha} stability in ARPE-19 cells. Confluent ARPE-19 cells were serum starved for 24 h before treatment with 100 nM IGF-I or 100 µM CoCl2 in fresh serum-free medium. After a 4-h incubation, cells were lysed and protein concentrations were determined. Lysates (100 µg) were solubilized in SDS sample buffer, reduced with DTT, resolved on a 12.5% acrylamide gel, transferred to nitrocellulose, and probed for HIF-1{alpha} (A) and HIF-1{beta} (B). Bands were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies and the ECL reagent. Results shown are representative of 3 or more experiments. C: relative intensity of HIF-1{alpha} in relation to HIF-1{beta} control for each treatment. D: after transfection with p2.1 luciferase and SV40 Renilla for 24 h in serum-free medium, ARPE-19 cells were incubated for 18 h with 100 nM IGF-I or 100 µM CoCl2. Lysates were processed for dual luciferase (Luc) activity. Relative luciferase activity was calculated by dividing p2.1 luciferase by SV40 Renilla activity. Error bars represent SD between triplicate wells. Significant differences in HIF-1 reporter activity were observed (*P < 0.01, **P < 0.001). The plot is representative of 3 or more experiments.

 
To determine the temporal parameters of IGF-I-induced HIF-1{alpha} protein expression, cells were stimulated with 100 nM IGF-I over a 24-h time course. HIF-1{alpha} expression peaked between 4 and 6 h of stimulation and declined thereafter (Fig. 2). HIF-1{alpha} levels increased again at 24 h, although this change was not statistically significant (P > 0.05) compared with time 0. Treatment of serum-starved ARPE-19 cells with IGF-I for 4 h resulted in a dose-dependent increase in HIF-1{alpha} protein levels, with the maximal effect occurring at 10 nM. This increase was not significantly (P > 0.05) greater than the effect of 100 nM IGF-I (Fig. 3).



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Fig. 2. Time course of IGF-I-induced HIF-1{alpha} protein expression in ARPE-19 cells. Top: duplicate dishes of confluent ARPE-19 cells were serum starved for 24 h before treatment with 100 nM IGF-I in fresh serum-free medium for an additional 0–24 h. At the times indicated, cells were lysed and protein contents were determined. Lysates (100 µg) were solubilized in SDS sample buffer and analyzed for HIF-1{alpha} and {beta}-actin as detailed in Fig. 1. This blot is representative of 3 experiments. Bottom: densitometric normalization of relative HIF-1{alpha} intensity to {beta}-actin control for each treatment. Error bars represent SD between duplicate wells. Significant differences in HIF-1{alpha} protein expression were observed (*P < 0.075, **P < 0.05).

 


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Fig. 3. Dose-dependent effect of IGF-I on HIF-1{alpha} protein expression in ARPE-19 cells. Top: duplicate dishes of confluent ARPE-19 cells were serum starved for 24 h before treatment with a battery of IGF-I doses (10 pM-1 µM) in fresh serum-free medium. After 4 h of incubation, cell lysates were analyzed for HIF-1{alpha} and {beta}-actin as described in Fig. 1. This blot is representative of 3 experiments. Bottom: densitometric normalization of relative HIF-1{alpha} intensity to {beta}-actin control for each treatment. Error bars represent SD between duplicate wells. Significant differences in HIF-1{alpha} protein expression were observed (*P < 0.05, **P < 0.005).

 
VEGF and IGFBP-3 secretion. To examine VEGF secretion, serum-starved ARPE-19 cells were incubated in the presence or absence of 100 nM IGF-I for 1–24 h. As shown in Fig. 4A, immunoblot analysis of conditioned medium revealed the presence of a VEGF doublet of ~42 and 44 kDa. These represent VEGF homodimers that are singly and doubly glycosylated at each of the single consensus N-linked glycosylation sites per monomer. Time course studies indicated that VEGF accumulation exhibited a lag phase of ~4 h, with a significant level of growth factor accumulation in the medium appearing thereafter (Fig. 4B).



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Fig. 4. Time course of IGF-I-stimulated vascular endothelial growth factor (VEGF) secretion by ARPE-19 cells. A: confluent ARPE-19 cells grown in 6-well plates were serum starved for 24 h before treatment with 100 nM IGF-I or BSA carrier alone in fresh serum-free medium. After 12 h, conditioned medium from each well was processed for immunoblot analysis. B: duplicate 6-well plates of confluent ARPE-19 cells were serum starved for 24 h before treatment with 100 nM IGF-I or BSA carrier alone, in fresh serum free medium, for incubations ranging from 1 to 24 h. At each time point, conditioned medium was processed for immunoblot and densitometric quantification. Error bars represent SD in secretion between duplicate wells. Significant differences in VEGF secretion at indicated time points were observed (*P < 0.05). Data shown are representative of 3 or more experiments.

 
IGF-I was shown previously to stimulate the secretion of IGFBP-3 in various retinal cells including RPE cells (36, 41, 49). Thus we next examined IGFBP-3 secretion by serum-starved ARPE-19 cells incubated in the absence or presence of 100 nM IGF-I for 1–24 h. As shown in Fig. 5A, four bands ranging in size from ~28 to ~45 kDa were identified in conditioned medium by ligand blot analysis. Immunoblot analysis confirmed that these bands were all IGFBP-3 related (not shown). IGFBP-3 contains three consensus sites for N-linked glycosylation, explaining the observed banding pattern. To confirm this, ARPE-19 cells were incubated with tunicamycin, an inhibitor of N-linked sugar transfer from dolichol precursors (55). As shown in Fig. 5A, tunicamycin treatment of unstimulated or IGF-I-stimulated cells after a 2-h tunicamycin pretreatment resulted in the expression of a single species of IGFBP-3 in conditioned medium that comigrated with recombinant nonglycosylated IGFBP-3. Bands representing mono-, di-, and triglycosylated IGFBP-3 were absent. IGFBP-1, -2, -4, -5, and -6 were not detected by ligand or immunoblot. Yang and Chaum (64) similarly observed IGFBP-1, -2, and -4 to be nearly absent, whereas expression of IGFBP-3, -5, and -6 tended to vary in an RPE cell line-dependent manner. Significantly, we have observed similar profiles for the secretion of VEGF and IGFBP-3, along with the absence of other IGFBPs, in the RPE cell line D407 (49). Bands representing non-, mono-, di-, and triglycosylated IGFBP-3 were all included when quantifying IGFBP-3 secretion. As shown in Fig. 5B, secreted IGFBP-3 was not detectable in the medium for 4 h of incubation. From that point, accumulation in the medium was approximately linear. IGF-I addition significantly increased this level of IGFBP-3 secretion over that in unstimulated cells.



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Fig. 5. Time course of IGF-I-induced IGF binding protein (IGFBP)-3 secretion in cells. A: confluent ARPE-19 cells grown in 6-well plates were serum starved for 24 h before treatment in fresh serum-free medium with 100 nM IGF-I, 1 µg/ml tunicamycin, or BSA carrier alone after 2-h pretreatment with fresh serum-free medium alone or containing tunicamycin. After 12 h, conditioned medium from each well was processed for ligand blot analysis. B: duplicate 6-well plates of confluent ARPE-19 cells were serum starved for 24 h before treatment with 100 nM IGF-I or BSA carrier alone for incubations ranging from 1 to 24 h. At each time point, conditioned medium was processed for ligand blot and densitometric quantification. Error bars represent SD in secretion between duplicate wells. Significant differences in IGFBP-3 secretion at indicated time points were observed (*P < 0.05, **P < 0.005). Data shown are representative of 3 or more experiments.

 
As shown in Fig. 6, stimulation of ARPE-19 cells with a battery of IGF-I doses for 12 h caused a dose-dependent increase in VEGF secretion. Maximal secretion was obtained with 10 nM IGF-I, although statistically insignificant (P > 0.05) from the effect at 100 nM. Secretion at 100 nM, the IGF-I dose used in all time course studies, represents a 17-fold induction of VEGF secretion over control. Similar to VEGF, IGF-I stimulated a dose-dependent increase in IGFBP-3 secretion (Fig. 7). Again, maximal secretion was obtained with 10 nM IGF-I, although statistically insignificant (P > 0.05) from the effect at 100 nM. Secretion at 100 nM IGF-I elicited a 30-fold increase in IGFBP-3 secretion over control.



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Fig. 6. IGF-I dose-dependent increase in VEGF secretion by ARPE-19 cells. Duplicate 6-well plates of confluent ARPE-19 cells were serum starved for 24 h before treatment with increasing concentrations of IGF-I ranging from 10 pM to 1 µM in fresh serum-free medium. After a 12-h incubation, conditioned medium was subjected to immunoblot and densitometric quantification. Error bars represent SD in secretion between duplicate wells of ARPE-19 cells. Significant differences in VEGF secretion were observed (*P < 0.05, **P < 0.005). Densitometrically quantified immunoblots are representative of 3 experiments.

 


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Fig. 7. IGF-I dose-dependent increase in IGFBP-3 secretion by ARPE-19 cells. Conditioned medium was analyzed as in Fig. 8. Error bars represent SD in secretion between duplicate wells of ARPE-19 cells. Significant differences in IGFBP-3 secretion were observed (*P < 0.05, **P < 0.005). Densitometrically quantified ligand blots are representative of 3 experiments.

 
Autocrine relationship between VEGF and IGFBP-3. To examine whether VEGF secretion by ARPE-19 cells affects IGFBP-3 release, we added rhVEGF at two doses in an IGF-I dose-response assay. As illustrated in Fig. 8A, coaddition of 1 or 10 ng/ml rhVEGF in the presence or absence of IGF-I to ARPE-19 cells had no significant effect on HIF-1{alpha} protein levels. In contrast, coaddition of 1 ng/ml rhVEGF to cells treated with 0.5 or 10 nM IGF-I led to increases in IGFBP-3 accumulation that were greater than each dose of IGF-I alone (Fig. 8B). A 10-fold increase in VEGF concentration had no additional effect on IGFBP-3 secretion.



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Fig. 8. VEGF stimulation of IGFBP-3 secretion. A: confluent ARPE-19 cells were serum starved for 24 h before treatment with various doses of IGF-I (100 pM-10 nM) and recombinant human (rh)VEGF (1–10 ng/ml) in fresh serum-free medium. After 4 h of incubation, cells were lysed, lysates were processed for immunoblot as described in Fig. 1, and blots were probed for HIF-1{alpha}, HIF-1{beta}, and {beta}-actin. B: duplicate 6-well plates of confluent ARPE-19 cells were serum starved for 24 h before treatment with increasing concentrations of IGF-I ranging from 100 pM to 10 nM and either 1 ng/ml or 10 ng/ml rhVEGF in fresh serum-free medium. After a 12-h incubation, conditioned medium was subjected to immunoblot and densitometric quantification. Error bars represent SD in secretion between duplicate wells of ARPE-19 cells. Significant differences in IGFBP-3 secretion at indicated time points were observed (*P < 0.05, **P < 0.005). Data shown are representative of 3 experiments.

 
Treatment of ARPE-19 cells with rhIGFBP-3 alone had no effect on VEGF secretion. However, coaddition of rhIGFBP-3 with IGF-I resulted in a dose-dependent decrease in IGF-I-stimulated VEGF secretion (Fig. 9). IGF-I-stimulated VEGF secretion was reduced to control levels with 10 nM rhIGFBP-3 in cells treated with 0.5 nM IGF-I, whereas 100 nM IGFBP-3 was required in cells treated with 10 nM IGF-I. Addition of 10 nM rhIGFBP-3 to cells treated with 10 nM IGF-I led to an ~60% reduction in VEGF secretion, approximately equivalent to the secretory response observed with the 0.5 nM IGF-I dose alone.



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Fig. 9. IGFBP-3 antagonism of IGF-I-induced VEGF secretion. Duplicate 6-well plates of confluent ARPE-19 cells were serum starved for 24 h before treatment with increasing concentrations of IGF-I (100 pM to 10 nM) and either 10 nM or 100 nM rhIGFBP-3 in fresh serum-free medium. After a 12-h incubation, conditioned medium was subjected to immunoblot and densitometric quantification. Error bars represent SD in secretion between duplicate wells of ARPE-19 cells. Significant differences in VEGF secretion at indicated time points were observed (*P < 0.05, **P < 0.01). This plot is representative of 3 experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
IGF-I stimulated a time- and dose-dependent increase in HIF-1{alpha} protein, the regulated member of the HIF-1 heterodimer. Luciferase reporter assays of HIF-1 transcriptional activity demonstrated accumulation of HIF-1{alpha} correlated with the formation of functional, HRE-binding, HIF-1 heterodimers. Interestingly, time courses revealed IGF-I stimulation of HIF-1{alpha} peaking at 4–6 h, with a second increase in HIF-1{alpha} sporadically occurring at 24 h. Although statistically insignificant, the potential autocrine effect of IGF-I-induced secretion and accumulation of cytokines and/or growth factors in the conditioned medium on the expression of HIF-1{alpha} protein has yet to be examined.

Although the signaling cascade leading to IGF-I-induced HIF-1{alpha} expression is still intensely debated, it is well established that the VEGF promoter contains HREs, activation of which results from the binding of HIF-1 (reviewed in Ref. 45). Similarly, a connection, although tenuous, has been established between HIF-1 activity and IGFBP-3 protein expression. Work by Feldser and colleagues (16) demonstrated that although the IGFBP-3 promoter lacks an obvious HRE, IGFBP-3 gene expression was markedly reduced in HIF-1{alpha}-deficient cells under hypoxic conditions. These findings suggest some other level of regulation, possibly an indirect effect. In addition, there is still controversy in the literature as to whether IGF-I also upregulates the translation of HIF-1{alpha}. Irrespective of the mechanisms by which HIF-1 levels are increased, the present results demonstrate that IGF-I stimulates VEGF and IGFBP-3 secretion in a time- and dose-dependent manner. Together, these findings extend the initial work of Randolph et al. (41) and Punglia et al. (40) demonstrating IGF-I-induced increases in VEGF and IGFBP-3, respectively.

Elevated subretinal levels of VEGF can act to trigger the progression of CNV in animal models (4, 24, 27, 50, 62). VEGF and its receptors, including Flk-1 and feline sarcoma virus-like tyrosine receptor-1 (Flt-1), colocalize to RPE cells (8, 21). Accordingly, we examined whether ARPE-19 cells respond to VEGF. Whereas rhVEGF addition did not alter HIF-1{alpha} expression, it did stimulate secretion of IGFBP-3, suggesting that VEGF may regulate RPE cell function in an autocrine manner.

Low oxygen tension (1–2%) significantly promotes angiogenesis by stimulating VEGF secretion and the upregulation of KDR (7, 61). These conditions also promote the formation of oxygen radicals through a mechanism involving the electron transport chain. It has been reported that reactive oxygen species (ROS) increase the DNA binding activity of HIF-1{alpha} (7). It is tempting to speculate that VEGF stimulation of ROS leads to greater HIF-1 binding to the HRE in the IGFBP-3 promoter, leading to increased expression of IGFBP-3. This may serve to explain the observed VEGF stimulation of IGFBP-3 secretion in the absence of detectable alterations in HIF-1{alpha} expression.

IGFBP-3 release by RPE cells may have important implications in the regulation of IGF-I/IGF-II autocrine and/or paracrine functions at the RPE and photoreceptor layers, given that IGF action may be inhibited (42) or enhanced (5, 13) by IGFBP-3. Our results demonstrate that IGF-I stimulates IGFBP-3 secretion in a time- and dose-dependent manner. Furthermore, IGF-I-induced VEGF secretion was attenuated by rhIGFBP-3 addition. In light of the fact that IGF-I upregulates the secretion of IGFBP-3 and VEGF in ARPE-19 cells and that their secretion occurs at the apical pole in polarized RPE cells (33, 49), the ability of IGFBP-3 to reduce the bioavailability of IGF-I may play a major role in modulating VEGF secretion by RPE cells in the subretinal space (25, 33, 49). As such, fluctuations in IGF-I, IGF-II, or IGFBPs may have significant implications on RPE cell proliferation and migration after choroidal capillary invasion and the subsequent leakage of circulatory IGFs from choroidal vessels (41, 48, 52, 58, 66). Consequently, dysregulation of the IGF-I system at the level of the subretina may contribute to changes in RPE morphology and increases in angiogenic factor secretion, consistent with CNV.

In summary, we have shown that IGF-I stimulates the expression of HIF-1{alpha} and the formation of functional HIF-1 dimers as well as the secretion of VEGF and IGFBP-3 in a time- and dose-dependent manner. In contrast, VEGF enhances the secretion of IGFBP-3 both in the absence and presence of IGF-I without affecting HIF-1{alpha} protein expression. Although it had no effect alone, IGFBP-3 attenuated IGF-I-induced VEGF secretion to control levels when present in 10-fold molar excess of exogenously added IGF-I. Together, these results provide further evidence for a role of an IGF-I autocrine/paracrine system in the retina, both in terms of normal ocular physiology as well as in the progression of CNV. The ability of rhVEGF to enhance IGFBP-3 expression, which in turn attenuates IGF-I-stimulated VEGF secretion, constitutes a novel negative autocrine loop regulating this potent angiogenic factor. Furthermore, the ability of rhIGFBP-3 to attenuate IGF-I stimulation of VEGF to constitutive levels presents a tempting avenue in the development of peptide mimetics that retain the IGF-I antagonistic properties of IGFBP-3. Such antagonists may be helpful in the treatment of a wide variety of ischemic retinal disorders linked to neovascularization of the retina and iris where serum and vitreous IGF-I levels are elevated (40). Although HIF-1{alpha} is primarily maintained at low levels under normoxic conditions by a degradation process involving the ubiquitin-proteasome system, several cytokines have been found to increase HIF-1 activity (17, 22, 35, 43, 45, 57). Van Obberghen and colleagues (57) reported that insulin stimulates HIF-1{alpha} translation via a phosphatidylinositol 3-kinase (PI3-kinase)-dependent signaling pathway in ARPE-19 cells. They also reported that insulin and IGF-I stimulate VEGF expression via different signaling pathways in NIH 3T3 cells (35). Whereas insulin stimulates PI 3-kinase/protein kinase B (PKB), induction by IGF-I involves ERK/mitogen-activated protein kinase (MAPK). In contrast, Semenza and colleagues (17) reported that IGF-I induces HIF-1{alpha} synthesis through both PI 3-kinase and MAPK pathways in HCT116 human colon cancer cells. We propose to carry out studies designed to elucidate the roles of reduced oxygen tension and retinal cytokines on HIF-1{alpha} expression and VEGF and IGFBP-3 secretion in the RPE and their influence on the progression of CNV. Studies at the cellular level will provide important insights into the mechanisms underlying the pathologies observed in the animal models of CNV. This will lead to a better understanding of the pathogenesis of this disease and to better treatments for this leading cause of blindness.


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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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This work was supported, in part, by National Cancer Institute Grant CA-78887 and Department of Defense Grant N6311601MD10004 to Hollings Cancer Center (to S. A. Rosenzweig).


    ACKNOWLEDGMENTS
 
We thank Dr. Robert G. Gourdie and the Gourdie lab for epifluorescence microscopy assistance.

A portion of this work was presented at the 85th Annual Meeting of the Endocrine Society, June 2002, San Francisco, CA; the 1st joint symposium of the Growth Hormone Research Society and the International Society for Insulin-like Growth Factor Research, October 2002, Boston, MA; and the Association for Research in Vision and Ophthalmology Annual Meeting, May 2003, Fort Lauderdale, FL.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. A. Rosenzweig, Dept. of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical Univ. of South Carolina, 173 Ashley Ave., Charleston, SC 29425 (E-mail: rosenzsa{at}musc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Adamis AP, Shima DT, Tolentino M, Gragoudas ES, Ferrara N, Folkman J, D'Amore PA, and Miller JW. Inhibition of VEGF prevents retinal ischemia-associated iris neovascularization in a primate. Arch Ophthalmol 114: 66–71, 1996.[Abstract]

2. Aiello LP, Pierce EA, Foley ED, Takagi H, Chen H, Riddle L, Ferrara N, King G, and Smith LEH. Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proc Natl Acad Sci USA 92: 10457–10461, 1995.[Abstract]

3. Amin RH, Frank RN, Eliot D, and Puklin JE. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) immunoreactivity in human choroidal neovascular membranes (Abstract). Invest Ophthalmol Vis Sci 36: S552, 1995.

4. Amin RH, Puklin JE, and Frank RN. Growth factor localization in choroidal neovascular membranes of age-related macular degenerations. Invest Ophthalmol Vis Sci 35: 3178–3188, 1994.[Abstract]

5. Blum WF, Jenne EW, Reppin F, Kietzmann K, Ranke MB, and Bierich JR. Insulin-like growth factor I (IGF-I)-binding protein complex is a better mitogen than free IGF-I. Endocrinology 125: 766–772, 1989.[Abstract]

6. Campochiaro PA, Jerdan JA, and Glaser BM. The extracellular matrix of human retinal pigmented epithelial cells in vivo and its synthesis in vivo. Invest Ophthalmol Vis Sci 27: 1615–1621, 1986.[Abstract]

7. Chandel NS, Maltepe E, Goldwasser E, Mathieu CE, Simon MC, and Schumacker TP. Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc Natl Acad Sci USA 95: 11715–11720, 1998.[Abstract/Free Full Text]

8. Chen YS, Hackett SF, Schoenfeld CL, Vinores MA, Vinores SA, and Campochiaro PA. Localization of vascular endothelial growth factor and its receptors to cells of vascular and avascular epiretinal membranes. Br J Ophthalmol 81: 919–926, 1997.[Abstract/Free Full Text]

9. Cui JZ, Kimura H, Spee C, Thumann G, Hinton DR, and Ryan SJ. Natural history of choroidal neovascularization induced by vascular endothelial growth factor in the primate. Graefes Arch Clin Exp Ophthalmol 238: 326–333, 2000.[CrossRef][ISI][Medline]

10. D'Amato RJ and Adamis AP. Angiogenesis inhibition in age-related macular degeneration. Ophthalmology 102: 1261–1262.

11. Dawson DW, Volpert OV, Gillis P, Crawford SE, Xu H, Benedict W, and Bouck NP. Pigment epithelium derived factor: a potent inhibitor of angiogenesis. Science 285: 245–248, 1999.[Abstract/Free Full Text]

12. Dunn KC, Aotaki-Keen AE, Putkey FR, and Hjelmeland LM. ARPE-19, a human retinal pigment epithelial cell line with differentiated properties. Exp Eye Res 62: 155–169, 1996.[CrossRef][ISI][Medline]

13. Elgin RG, Busby WH Jr, and Clemmons DR. An insulin-like growth factor (IGF) binding protein enhances the biological response to IGF-I. Proc Natl Acad Sci USA 84: 3254–3258, 1987.[Abstract]

14. Epstein AC, Gleadle JM, McNeill LA, Hewitson KS, O'Rourke J, Mole DR, Mukherji M, Metzen E, Wilson MI, Dhanda A, Tian YM, Masson N, Hamilton DL, Jaakkola P, Barstead R, Hodgkin J, Maxwell PH, Pugh CW, Schofield CJ, and Ratcliffe PJ. C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 107: 43–54, 2001.[ISI][Medline]

15. Feldman EL and Randolph AE. Regulation of insulin-like growth factor binding protein synthesis and secretion in human retinal pigment epithelial cells. J Cell Physiol 158: 198–204, 1994.[ISI][Medline]

16. Feldser D, Agani F, Iyer NV, Pak B, Ferreira G, and Semenza GL. Reciprocal positive regulation of hypoxia-inducible factor 1{alpha} and insulin-like growth factor 2. Cancer Res 59: 3915–3918, 1999.[Abstract/Free Full Text]

17. Fukuda R, Hirota K, Fan F, Jung YD, Ellis LM, and Semenza GL. IGF-1 induces HIF-1-mediated VEGF expression that is dependent on MAP kinase and PI-3-kinase signaling in colon cancer cells. J Biol Chem 277: 38205–38211, 2002.[Abstract/Free Full Text]

18. Green WR. Clinicopathologic studies of treated choroidal neovascular membranes. A review and report of two cases. Retina 11: 328–356, 1991.[ISI][Medline]

19. Green WR. The retina. In: Ophthalmic Pathology: an Atlas and Textbook. Philadelphia, PA: Saunders, 1996, chapt. 9, p. 982–1051.

20. Green WR and Key SN. Senile macular degeneration: a histopathologic study. Trans Am Ophthalmol Soc 75: 180–254, 1977.[Medline]

21. Guerrin M, Moukadiri H, Chollet P, Moro F, Dutt K, Malecaze F, and Plouet J. Vasculotropin/vascular endothelial growth factor for human retinal pigment epithelial cells cultured in vitro. J Cell Physiol 164: 385–394, 1995.[ISI][Medline]

22. Huang LE, Gu J, Schau M, and Bunn HF. Regulation of hypoxia-inducible factor 1{alpha} is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Proc Natl Acad Sci USA 95: 7987–7992, 1998.[Abstract/Free Full Text]

23. Husain D, Kramer M, Kenny AB, Michaud N, Flotte TH, Gragoudas ES, and Miller JW. Effects of photodynamic therapy using verteporfin on experimental choroidal neovascularization and normal retina and choroid up to 7 weeks after treatment. Invest Ophthalmol Vis Sci 40: 2322–2331, 1999.[Abstract/Free Full Text]

24. Ishibashi T, Hata Y, Yoshikawa H, Sueishi K, and Inomata H. Expression of vascular endothelial growth factor in experimental choroidal neovascularization. Graefes Arch Clin Exp Ophthalmol 235: 159–167, 1997.[ISI][Medline]

25. Jones JI and Clemmons DR. Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev 16: 3–34, 1995.[ISI][Medline]

26. Keck PJ, Hauser SD, Krivi G, Sanzo K, Warren T, Feder J, and Connolly DT. Vascular permeability factor, an endothelial cell mitogen related to PDGF. Science 246: 1309–1312, 1989.[ISI][Medline]

27. Krzystolik MG, Afshari MA, Adamis AP, Gaudreault J, Gragoudas ES, Michaud NA, Li W, Connolly E, O'Neil CA, and Miller JW. Prevention of experimental choroidal neovascularization with intravitreal anti-vascular endothelial growth factor antibody fragment. Arch Ophthalmol 120: 338–346, 2002.[Abstract/Free Full Text]

28. Kvanta A, Algvere PV, Berglin L, and Seregard S. Subfoveal fibrovascular membranes in age-related macular degeneration express vascular endothelial growth factor. Invest Ophthalmol Vis Sci 37: 1929–1934, 1996.[Abstract]

29. Leung DW, Cachianes G, Kuang WJ, Goeddel DV, and Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 246: 1306–1309, 1989.[ISI][Medline]

30. Lopez PF, Sippy BD, Lamber HM, Thach AB, and Hinton DR. Transdifferentiated retinal pigment epithelial cells are immunoreactive for vascular endothelial growth factor in surgically excised age-related macular degeneration-related choroidal neovascular membranes. Invest Ophthalmol Vis Sci 37: 855–868, 1996.[Abstract]

31. The Macular Photocoagulation Study Group. Argon laser photocoagulation for senile macular degeneration: results of a randomized clinical trial. Arch Ophthalmol 100: 912–918, 1982.[Abstract]

32. The Macular Photocoagulation Study Group. Argon laser photocoagulation for neovascular maculopathy: five year results from randomized clinical trials. Arch Ophthalmol 109: 1109–1114, 1991.[Abstract]

33. Marmorstein AD, Csaky KG, Baffi J, Lam L, Rahaal F, and Rodriguez-Boulan E. Saturation of, and competition for entry into, the apical secretory pathway. Proc Natl Acad Sci USA 97: 3248–3253, 2000.[Abstract/Free Full Text]

34. Martin DM, Yee D, and Feldman EL. 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, 1992.[ISI][Medline]

35. Miele C, Rochford JJ, Filippa N, Giorgetti-Peraldi S, and Van Obberghen E. Insulin and insulin-like growth factor-1 induce vascular endothelial growth factor mRNA expression via different signaling pathways. J Biol Chem 275: 21695–21702, 2000.[Abstract/Free Full Text]

36. Moriarty P, Boulton M, Dickson A, and McLeod D. Production of IGF-1 and IGF binding proteins by retinal cells in vitro. Br J Ophthalmol 78: 638–642, 1994.[Abstract]

37. Mousa SA, Lorelli W, and Campochiaro PA. Role of hypoxia and extracellular matrix-integrin binding in the modulation of angiogenic growth factors secretion by retinal pigment epithelial cells. J Cell Biochem 74: 135–143, 1999.[CrossRef][ISI][Medline]

38. Ocrant I, Fay CT, and Parmelee JT. Expression of insulin and insulin-like growth factor receptors and binding proteins by retinal pigment epithelium. Exp Eye Res 52: 581–589, 1991.[CrossRef][ISI][Medline]

39. Ozaki H, Yu AY, Della N, Ozki K, Luna JD, Yamada H, Hackett SF, Okamoto N, Zack DJ, Semenza GL, and Campochiaro PA. Hypoxia inducible factor 1{alpha} is increased in ischemic retina: temporal and spatial correlations with VEGF expression. Invest Ophthalmol Vis Sci 40: 182–188, 1999.[Abstract]

40. Punglia RS, Lu M, Hsu J, Kuroki M, Tolentino J, Keough K, Levy AP, Levy NS, Goldberg MA, D'Amato RJ, and Adamis AP. Regulation of VEGF expression by IGF-1. Diabetes 46: 1619–1626, 1997.[Abstract]

41. Randolph A, Yee D, and Feldman EL. Insulin-like growth factor binding protein expression in human retinal pigment epithelial cells. Ann NY Acad Sci 692: 265–267, 1993.[ISI][Medline]

42. Rutanen EM, Pekonen F, and Makinen T. Soluble 34K binding protein inhibits the binding of insulin-like growth factor I to its cell receptors in human secretory phase endometrium: evidence for autocrine/paracrine regulation of growth factor action. J Clin Endocrinol Metab 66: 173–180, 1988.[Abstract]

43. Salceda S and Caro J. Hypoxia-inducible factor 1{alpha} (HIF-1{alpha}) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. J Biol Chem 272: 22642–22647, 1997.[Abstract/Free Full Text]

44. Semenza GL. Expression of hypoxia inducible factor 1: mechanisms and consequences. Biochem Pharmacol 59: 47–53, 1999.[CrossRef][ISI]

45. Semenza GL. Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1. Annu Rev Cell Dev Biol 15: 551–578, 1999.[CrossRef][ISI][Medline]

46. Semenza GL, Jiang BH, Leung SW, Passantino R, Concordet JP, Maire P, and Giallongo A. Hypoxia response elements in the aldolase A, enolase 1, and lactate dehydrogenase A gene promoters contain essential binding sites for hypoxia-inducible factor 1. J Biol Chem 271: 32529–32537, 1996.[Abstract/Free Full Text]

47. Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, and Dvorak HF. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 219: 983–985, 1983.[ISI][Medline]

48. Seregard S, Algvere PV, and Berglin L. Immunohistochemical characterization of surgically removed subfoveal fibrovascular membranes. Graefes Arch Clin Exp Ophthalmol 232: 325–329, 1994.[ISI][Medline]

49. Slomiany MG and Rosenzweig SA. IGF-1 induced VEGF and IGFBP-3 secretion correlates with increased HIF-1{alpha} expression and activity in the retinal pigment epithelial cell line D407. Invest Ophthalmol Vis Sci. In press.

50. Spilsbury K, Garrett KL, Shen WY, Constable IJ, and Rakoczy PE. Overexpression of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium leads to the development of choroidal neovascularization. Am J Pathol 157: 135–144, 2000.[Abstract/Free Full Text]

51. Spinola SM and Cannon JG. Different blocking agents cause variations in the immunological detection of proteins transferred to nitrocellulose membranes. J Immunol Methods 81: 161–165, 1985.[CrossRef][ISI][Medline]

52. Spraul CW, Kaven C, Amann J, Lang GK, and Lang GE. 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, 2000.[CrossRef][ISI][Medline]

53. Takagi H, Yoshimura N, Tanihara H, and Honda Y. Insulin-like growth factor-related genes, receptors, and binding proteins in cultured human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 35: 916–923, 1994.[Abstract]

54. Tarnowski BI, Shepherd V, and McLaughlin BJ. Mannose-6-phosphate receptors on the plasma membrane of rat retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 29: 291–297, 1988.[Abstract]

55. Tkacz JS and Lampen O. Tunicamycin inhibition of polyisoprenyl N-acetylglucosaminyl pyrophosphate formation in calf-liver microsomes. Biochem Biophys Res Commun 65: 248–257, 1975.[ISI][Medline]

56. Tombran-Tink J, Chader GJ, and Johnson LV. PEDF: a pigment epithelium-derived factor with potent neuronal differentiative activity. Exp Eye Res 53: 411–414, 1991.[ISI][Medline]

57. Treins C, Giorgetti-Peraldi S, Murdaca J, Semenza GL, and Van Obberghen E. Insulin stimulates hypoxia-inducible factor 1 through a phosphatidylinositol 3-kinase/target of rapamycin-dependent signaling pathway. J Biol Chem 277: 27975–27981, 2002.[Abstract/Free Full Text]

58. Wada M, Ogata N, Otsuji T, and Uyama M. Expression of vascular endothelial growth factor and its receptor (KDR/flk-1) mRNA in experimental choroidal neovascularization. Curr Eye Res 18: 203–213, 1999.[CrossRef][ISI][Medline]

59. Waldbillig RJ, Fletcher RT, Somers RL, and Chader GJ. IGF-1 receptors in the bovine neural retina: structure, kinase activity, and comparison with retinal insulin receptors. Exp Eye Res 47: 587–607, 1988.[ISI][Medline]

60. Waldbillig RJ, Pfeffer BA, Schoen TJ, Adler AA, Shen-Orr Z, Scavo L, LeRoith D, and Chader GJ. Evidence for an insulin-like growth factor autocrine-paracrine system in the retinal photoreceptor pigment epithelial cell complex. J Neurochem 57: 1522–1533, 1991.[ISI][Medline]

61. Waltenberger J, Mayr U, Pentz S, and Hombach V. Functional upregulation of the vascular endothelial growth factor receptor KDR by hypoxia. Circulation 94: 1647–1654, 1996.[Abstract/Free Full Text]

62. Wells JA, Murthy R, Chibber R, Nunn A, Molinatti PA, Kohner EM, and Gergor ZJ. Levels of vascular endothelial growth factor are elevated in the vitreous of patients with subretinal neovascularization. Br J Ophthalmol 80: 363–366, 1996.[Abstract]

63. Wistow G, Bernstein SL, Wyatt MK, Fariss RN, Behal A, Touchman JW, Bouffard G, Smith D, and Peterson K. Expressed sequence tag analysis of human RPE/choroid for the NEIBank Project: over 6000 non-redundant transcripts, novel genes, and splice variants. Mol Vis 8: 205–220, 2002.[ISI][Medline]

64. Yang H and Chaum E. A reassessment of insulin-like growth factor binding protein gene expression in the human retinal pigment epithelium. J Cell Biochem 89: 933–943, 2003.[CrossRef][ISI][Medline]

65. Yi X, Ogata N, Komada M, Takahashi K, Omori K, and Uyama M. Vascular endothelial growth factor expression in choroidal neovascularization in rats. Graefes Arch Clin Exp Ophthalmol 235: 313–319, 1997.[ISI][Medline]

66. Zelzer E, Levy Y, Kahana C, Shilo BZ, Rubinstein M, and Cohen B. Insulin induces transcription of target genes through the hypoxia-inducible factor HIF-1{alpha}/ARNT. EMBO J 17: 5085–5094, 1998.[Abstract/Free Full Text]

67. Zick Y, Spiegel AM, and Sagi-Eisenberg R. Insulin-like growth factor 1 receptors in retinal rod outer segments. J Biol Chem 262: 10259–10264, 1987.[Abstract/Free Full Text]

68. Zinn KM and Benjamin-Henkind JV. Anatomy of the human retinal pigment epithelium. In: The Retinal Pigment Epithelium, edited by Zinn KM and Marmor MF. Cambridge, MA: Harvard Univ. Press, 1979, chapt. 1, p. 3–31.